Isolation and characterization of the high-affinity K(+)-translocating ATPase from Rhodobacter sphaeroides. Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K(+)-stimulated and Mg(2+)-stimulated ATPase was purified from membranes of these cells by solubilization with decyl-beta-D-maltoside in the presence of Escherichia coli phospholipids followed by triazine-dye affinity chromatography. This primary transport system has a substrate specificity and an inhibitor sensitivity closely similar to those of the Kdp ATPase from E. coli and is composed of three subunits with molecular masses of 70.0, 43.5, and 23.5 kDa.


Identification and molecular analysis of a 63-kilodalton stress protein from Neisseria gonorrhoeae. Iron limitation, glucose deprivation, and growth under low oxygen supply (environmental stress) increased the expression of several proteins of Neisseria gonorrhoeae, including a 63-kilodalton protein identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This gonococcal stress protein (GSP63) was detected in the cytosol and copurified with lithium acetate-derived outer membranes. Successful purification of the protein was achieved by sucrose density gradient centrifugation and by chromatography on phenyl-Sepharose. Gel filtration of the purified protein revealed a molecular weight of approximately 450,000, suggesting that in its native state, the protein consists of a multimer of six to eight subunits. Isoelectric focusing indicated a pI of 5.2. Immunoblotting experiments using a polyclonal antiserum raised against the purified protein demonstrated cross-reactivity with a protein of the same electrophoretic mobility as GSP63 in all eight gonococcal isolates tested. N-terminal amino acid sequencing of the protein revealed up to 65% homology with members of the Hsp60 heat shock protein family, suggesting that GSP63 is related to this group of proteins. This relationship was further substantiated by the immunological cross-reactivity of GSP63 with mycobacterial Hsp60 and the ATP-binding activity of the gonococcal stress protein.


Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity. Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed.


Cloning, nucleotide sequence, expression, and chromosomal location of ldh, the gene encoding L-(+)-lactate dehydrogenase, from Lactococcus lactis. A gene (designated ldh) that encodes fructose-1,6-bisphosphate-activated L-(+)-lactate dehydrogenase was cloned from Lactococcus lactis subsp. lactis. Plasmids containing ldh conferred fructose-1,6-bisphosphate-activated L-(+)-lactate dehydrogenase activity on Escherichia coli cells. This activity was conferred only when a promoter had been introduced into the plasmid to express the cloned ldh. The nucleotide sequence of ldh predicted a chain length of 324 amino acids and a subunit molecular weight of 34,910 for the enzyme, after removal of the N-terminal methionine residue. Northern analyses of L. lactis subsp. lactis RNA showed that a 4.1-kb transcript hybridized strongly with ldh and that 1.2- and 1.1-kb transcripts hybridized to much lesser extents. Promoter- and terminator-cloning studies in which we used the vectors pGKV210 and pGKV259 in L. lactis subsp. lactis revealed that the 5' flanking DNA of ldh is devoid of transcription initiation signals and that transcription entering the 3' flanking DNA from either direction is efficiently terminated. These data and the data from Northern analyses led to the conclusion that ldh is expressed as the 3' gene of the 4.1-kb transcript and suggested that posttranscriptional processing yielded the shorter transcripts. We determined that ldh is located on the L. lactis subsp. lactis chromosome between coordinates 1.619 and 1.669 of the previously reported physical map (D. L. Tulloch, L. R. Finch, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 173:2768-2775, 1991).


DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III. In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha.


The virulence gene activator ToxT from Vibrio cholerae is a member of the AraC family of transcriptional activators. Virulence gene expression in Vibrio cholerae is postulated to involve ToxR-dependent activation of the toxT gene followed by ToxT activation of virulence genes, including several of those involved in biogenesis of the toxin-coregulated pilus. ToxR is a transmembrane, DNA-binding protein which is a member of the OmpR subclass of two-component activator systems in bacteria. Data presented in this report demonstrate that ToxT is similar to the AraC family of transcriptional activators identified in a variety of gram-negative bacteria. The toxT open reading frame begins approximately 200 nucleotides from the end of the tcpF gene, which is part of a cluster of genes responsible for production of the toxin-coregulated pilus. Accumulation of toxT specific mRNA is ToxR dependent and is modulated by environmental conditions that modulate expression of the regulon. Within the intergenic region between tcpF and toxT is a potential stem-loop structure of an unusual nature which may play a role in regulating expression of toxT mRNA. Experiments with tcpF and toxT cloned behind a strong, constitutive promoter suggest that the two genes can be cotranscribed, but Northern (RNA) blot analysis of V. cholerae suggests that if they are, steady-state levels of their messages may be controlled by a posttranscriptional mechanism. Possible mechanisms for ToxR-dependent expression of toxT are discussed.


Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri. We have identified a new gene, glpX, belonging to the glp regulon of Escherichia coli, located directly downstream of the glpK gene. The transcription of glpX is inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gels. In addition to determining the E. coli glpX sequence, we also sequenced the corresponding glpFKX region originating from Shigella flexneri, which after transfer into E. coli was instrumental in elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C. Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in glpF. The most striking difference between the E. coli and S. flexneri DNA was found directly behind glpK, where two repetitive (REP) sequences were present in S. flexneri, but not in the E. coli sequence. The presence or absence of these REP sequences had no effect on transport or on growth on glycerol. Not including the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced was different in the two sequences. Comparison of the sequence with those in the EMBL data library revealed a 99% identity between the last third of glpX and the first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the Escherichia coli chromosome and not, as reported, from the 7-min region and that the gene product identified as MvrA is in fact encoded by a gene distal to glpX.


Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization. In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast.


Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains. Plasmid pE194 has been shown to be rescued by integration after cultivation of infected Bacillus subtilis recE4 cells at a restrictive high temperature. The plasmid is also spontaneously excised from the chromosome at a low frequency by precise or imprecise excision (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). We have investigated nine excision plasmids, carrying insert DNA 1 to 6 kbp in length, either in a complete pE194 or in a partially deleted pE194 copy. Type 1 (additive) excision plasmids have the left- and right-junction DNAs preserved as 13-bp direct repeats (5'-GGGGAGAAAACAT-3') corresponding to the region between positions 864 and 876 in pE194. In type 2 (substitutive) excision plasmids, a conserved 13-bp sequence remains only at the right junction while the left junction has been deleted during the excision process. The type 3 excision plasmid carries at each junction the tetranucleotide 5'-TCCC-3', present in pE194 between positions 1995 and 1998. Although we isolated the excision plasmids from different integration mutants, the insert DNAs of eight independently isolated plasmids showed striking sequence homology, suggesting that they originated from one distinct region of the B. subtilis chromosome. Thus, we postulate that imprecise excision of pE194 occurs most frequently after its translocation from the original insertion site into a preferred excision site within the host chromosome. The imprecise excision from this site occurs at excision breakpoints outside the pE194-chromosome junctions in a chromosomal region which remains to be investigated further.


The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations. The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.


Molecular cloning, sequencing, and overexpression of the structural gene encoding the delta subunit of Escherichia coli DNA polymerase III holoenzyme. Using an oligonucleotide hybridization probe, we have mapped the structural gene for the delta subunit of Escherichia coli DNA polymerase III holoenzyme to 14.6 centisomes of the chromosome. This gene, designated holA, was cloned and sequenced. The sequence of holA matches precisely four amino acid sequences obtained for the amino terminus of delta and three internal tryptic peptides. A holA-overproducing plasmid that directs the expression of delta up to 4% of the soluble protein was constructed. Sequence analysis of holA revealed a 1,029-bp open reading frame that encodes a protein with a predicted molecular mass of 38,703 Da. holA may reside downstream of rlpB in an operon, perhaps representing yet another link between structural genes for the DNA polymerase III holoenzyme and proteins involved in membrane biogenesis. These and other features are discussed in terms of genetic regulation of delta-subunit synthesis.


Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein. VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.


Altered-function mutations of the transcriptional regulatory gene virG of Agrobacterium tumefaciens. Three point mutations were isolated in the Agrobacterium tumefaciens virG gene by screening for vir gene expression in the absence of added phenolic inducing compounds. All three mutations were localized in the predicted amino-terminal phosphoryl receiver domain of the protein. One mutant (N54D) bypasses the requirement for VirA and phenolic inducers both for transcriptional activation of all tested vir promoters and for plant tumorigenesis. This mutant also activates vir gene expression efficiently at neutral pH, indicating that the step in induction that is normally stimulated by acid pH occurs before or during VirG phosphorylation. The other two mutants (M13T and H15R) require VirA for activity but are sensitized to low levels of inducing stimuli.


Regulation of the Staphylococcus aureus plasmid pI258 mercury resistance operon. Experiments involving fusion between the Staphylococcus aureus plasmid pI258-encoded mer operon and the reporter gene beta-lactamase, mutational analysis, and trans-complementation studies have shown that the merR gene of pI258, which shows DNA sequence similarity with known merR genes from other bacteria, regulates the expression of the mer operon in vivo. The merR gene product is a trans-acting protein that activates mer operon transcription in the presence of the inducers Hg2+ and Cd2+. A glutathione-S-transferase-MerR fusion protein specifically bound and protected a 27-nucleotide operator sequence from DNase I digestion. This operator sequence is highly homologous with mer operator sequences of other known systems.


Influence of carbon source on cell surface topology of Thermomonospora curvata. The appearance of cell surface protuberances in Thermomonospora curvata correlated with cell-bound exoenzymes which could be removed by brief sonication. Mycelia grown on cellulose or xylan had numerous protuberances and retained 20 to 25% of endoglucanase and endoxylanase at cell surfaces, while those grown on pectin or starch had few protuberances and negligible bound pectinase or amylase.


Nucleotide sequence of the R721 shufflon. The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.


Resolution of chromosomes III and VI of Aspergillus nidulans by pulsed-field gel electrophoresis shows that the penicillin biosynthetic pathway genes pcbAB, pcbC, and penDE are clustered on chromosome VI (3.0 megabases). An improved electrophoretic molecular karyotype of Aspergillus nidulans ATCC 28901 has been obtained by contour-clamped electric field gel electrophoresis, which separates seven chromosomal bands and allows resolution of chromosomes III and VI. The three genes of the penicillin biosynthetic pathway, pcbAB, pcbC, and penDE, encoding alpha-aminoadipyl-cysteinyl-valine synthetase, isopenicillin N synthase, and isopenicillin N acyltransferase, respectively, are clustered together on a chromosome of 3.0 Mg, corresponding to linkage group VI, whereas the argB gene was located on a chromosome of 3.4 Mb, corresponding to linkage group III. Three other strains of A. nidulans contained a modified chromosome III of about 3.1 Mb that overlaps with chromosome VI, forming a doublet. Resolution of chromosomes III and VI in strain ATCC 28901 allowed unequivocal mapping of the penicillin gene cluster on chromosome VI of A. nidulans.


Crystallization and preliminary X-ray studies of a Bacillus subtilis and Thermus thermophilus HB8 chimeric 3-isopropylmalate dehydrogenase and thermostable mutants of it. A new type of chimeric 3-isopropylmalate dehydrogenase (2T2M6T) was produced by expressing the fused gene of Bacillus subtilis and Thermus thermophilus. The enzyme shows heat stability intermediate between those of the parents. The crystal of the enzyme belongs to the space group of P3(2)21, with cell dimensions of a = b = 78.9 A and c = 158.9 A. Two thermostable mutants of the chimeric enzyme were prepared by site-directed mutagenesis and then crystallized.


Tissue-specific regulation of renin-binding protein gene expression in rats. Rat gene for renin-binding protein (RnBP) was shown to be expressed in the kidney, adrenal gland, brain, lung, spleen, ovary, testis, and heart. On sodium depletion and captopril administration, the rat showed a marked increase in the adrenal RnBP mRNA level and a slight decrease in the kidney RnBP mRNA level. In two-kidney, one clip hypertensive rats, the RnBP mRNA levels of the clipped and contralateral kidneys were unchanged and also its adrenal mRNA level was maintained at the control level. The recombinant rat RnBP was synthesized in Escherichia coli cells and purified to apparent homogeneity. The RnBP existed as a homodimer and formed a heterodimer with rat renin to inhibit renin activity extensively. Intravenous injection of the RnBP into rats resulted in a rapid and strong inhibition of plasma renin activity, which persisted at least for 2 h. These results suggest that the expression of RnBP gene in the kidney and adrenal gland is regulated independently, and the function of RnBP is related to electrolyte homeostasis, probably through the interaction with renin.


Static and kinetic studies of calmodulin and melittin complex. Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.


Topological disposition of UDP-glucuronyltransferase in rat liver microsomes. The topological disposition of a form of UDP-glucuronyltransferase (called GT-1) in rat liver microsomes was examined. Concanavalin A-Sepharose failed to bind microsomal vesicles even though GT-1 has sugar chains of "high mannose" type, indicating that mannose-containing sugar chains of microsomal glycoproteins including GT-1 are not exposed to the outer surface of microsomal vesicles. Polyclonal antibodies raised against purified GT-1 could bind to microsomal vesicles, indicating that at least part of the GT-1 polypeptide chain is extruded to the outside of the microsomal membrane. Intact microsomal vesicles were digested with carboxypeptidase Y and then subjected to immunoblot analysis using the anti-GT-1 antibodies. It was thus found that the digestion resulted in cleavage of a C-terminal, 2-kDa fragment, leaving a 52-kDa fragment of GT-1 still tightly bound to the membrane. From these results, it is concluded that GT-1 is a transmembrane protein, which extrudes its C-terminal end (at least 2 kDa) to the outside of the membrane, whereas most of its polypeptide chain together with the sugar chains are located on the luminal side of the membrane.


Molecular cloning of a rat liver cDNA encoding the 16 kDa subunit of vacuolar H(+)-ATPases: organellar and tissue distribution of 16 kDa proteolipids. A cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of 15,807 Da (pI = 9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H(+)-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed.


Effect of diolein on hydrolysis of phosphatidylcholine by phospholipase C from Clostridium perfringens. The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.


Molecular cloning of a cDNA encoding a member of a novel cytochrome P450 family in the mollusc Lymnaea stagnalis. We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved home-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.


Purification and characterization of a novel dipeptidyl carboxypeptidase from a Streptomyces species. An extracellular protease derived from the culture broth of a microorganism, a Streptomyces species, produced Boc-Pro-Pro and diproline from Boc-Pro-Pro-Pro-Pro. The enzyme was purified 726-fold, with a yield of 2.6%, by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight of the enzyme was determined to be 65,000 by gel filtration and 70,000 by SDS-PAGE. The enzyme released a C-terminal dipeptide from peptide substrates having a C-terminal proline and a penultimate proline or alanine residue, but did not hydrolyze angiotensin I or bradykinin. When the enzyme hydrolyzed Leu-Pro-Pro-Pro-Pro-Pro, it produced Leu-Pro-Pro-Pro and Pro-Pro before producing Leu-Pro. The enzyme thus seems to be a kind of dipeptidyl carboxypeptidase, its substrate specificity being very different from that of the well known dipeptidyl carboxypeptidases [EC 3.4.15.1] such as the angiotensin-converting enzyme.


Leucine dehydrogenase from Bacillus stearothermophilus: identification of active-site lysine by modification with pyridoxal phosphate. We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.


Conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin. Pyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Man alpha 1-6(Man alpha 1-3)Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxy-succinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.


Twenty-eight-kilodalton phosphorylatable calcium- and lipid-binding proteins purified from Physarum plasmodium. Two 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0 microM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32 microM and the other of 3.2 mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0 microM and has a dissociation constant for lipid of 34 micrograms/ml at 10 microM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36 micrograms/ml for lipid at 2 mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.


A novel difucosylated neutral glycosphingolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus: I. Purification and structural determination of the glycolipid. A novel fucose-containing neutral glycosphingolipid (GL-5) was purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. The chemical structure was determined to be Fuc alpha 1-3GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-4Glc beta 1-1Cer by methylation analysis, partial acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The unique characteristics of GL-5 are that: the reducing terminal disaccharide portion is not Gal beta 1-4Glc but GlcNAc beta 1-4Glc; it includes a GalNAc beta 1-4GlcNAc sequence and a Fuc-GalNAc linkage; the defucosylated core is a novel trisaccharide chain; and the sugar structure is one of the smallest ever characterized for a difucosylated glycolipid. The major fatty acids were 22:1 and 22h:1, and about 30% of the total acids was 2-hydroxylated. All the long-chain bases were phytosphingosines, of which about 90% was n-t18:0. The similarity of the ceramide moiety to that of glucosylceramide from the same eggs [Kubo, H. et al. (1992) J. Biochem. 111, 726-731] suggests a close biosynthetic relationship between GL-5 and the glucosylceramide.


A novel difucosylated neutral glycosphingolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus: II. Structural determination by two-dimensional NMR. A novel fucosylglycolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus, was determined by using two-dimensional NMR methods. Subspectra extraction by the homonuclear Hartmann-Hahn method was useful for identification of the individual sugar components. The homonuclear Hartmann-Hahn and double-quantum-filtered correlated spectra were analyzed to establish the assignments of sugar proton resonances. On the basis of the resonance assignments, the linkages of the individual sugar components were determined to be as follows. [formula: see text] This glycolipid contains a novel skeletal structure with the linkages of GalNAc beta 1-4GlcNAc beta 1-4Glc beta. We also observed that 2-hydroxylation of the fatty acids induced appreciable chemical shift changes of the proton resonances of the phytosphingosine moiety as well as the anomeric proton resonance of the reducing terminal glucose.


General purpose medical imaging databases: three models. A useful general purpose digital medical image database project can begin with relatively simple and low-cost capabilities provided by a computer operating system. As the amount of data and the number of users grow, transition to a relational database model becomes appropriate. When multiple database systems are present, a flexible front end can provide sophisticated querying capabilities that bridge the systems, while hiding the complexities of the multiple systems from the user.


Update in biomedical visualization: the professional communicator's role. Health science communications professionals span a broad range of disciplines, each bringing a distinctly different focus and set of cognitive skills to bear upon information technologies. Nowhere is this more apparent than in biocommunications service units and academic programs. Until now, most faculty from visually-based disciplines were rarely required to generate research or project contracts. This is changing; new demands are being made for allied health faculty to obtain research grants and for academic health science centers to forge ventures with the private sector. Visualization in scientific computing offers new avenues for these activities.


Optical technology: making the simulator portable. Optical disc technology can be either analog or digital. Analog (videodiscs) simulation programs are available for the allied health professions educator to incorporate into the curriculum. Digital optical discs, currently available in three formats, follow standards that have been adopted by the International Standards Organization. The formats differ in the amount of information they can accommodate and their practical use in the health professions curriculum.


Instructional multimedia computing in the health sciences. This article focuses on the development and utilization of interactive videodisc (IVD) and multimedia instruction in the health sciences. The characteristics of IVD and multimedia are outlined and the four levels of IVD systems that can be used in health science education are described. The advantages of utilization of videodisc or multimedia materials are presented, as well as instructional approaches. Potential applications such as simulations, tutorials, role-modeling, and drill-and-practice are described. Research findings, levels of curricular integration, instructional delivery, and courseware networking are also described. The article concludes with suggestions for institutional development of IVD materials or the incorporation of off-the-shelf programs into health science curricula.


Partial characterization of the inhibitory effect of lipid peroxidation on the ouabain-insensitive Na-ATPase of rat kidney cortex plasma membranes. The present work evaluates the effect of lipid peroxidation on the ouabain-insensitive Na-ATPase of basolateral plasma membranes from rat kidney proximal tubular cells as an indirect way to study the lipid dependence of this enzyme. An inverse relationship between lipid peroxidation and Na-ATPase activity was found. This effect was due neither to a change in the optimal Km of the system for Na+ nor for the substrate Mg:ATP, nor the optimal pH value of the medium. The optimal temperature value, however, was shifted toward a higher value. There was also an increase of the apparent energy of activation in the region of temperatures above the transition point (20 degrees C) with increase in lipid peroxidation. Peroxidized membranes incubated with phosphatidylcholine from soybean restored their Na-ATPase activity. On the other hand, the Na-ATPase activity was sensitive to oleoly lysophosphatidylcholine. These results suggest that lipid peroxidation might be affecting the Na-ATPase activity through either an increase of peroxidized phospholipids, which might change the membrane fluidity of the lipid microenvironment of the ATPase molecules, or through a direct effect of lysophospholipids released during the lipid peroxidation.


Structure and properties of the coated vesicle (H+)-ATPase. Clathrin-coated vesicles play an important role in both receptor-mediated endocytosis and intracellular membrane traffic in eukaryotic cells. The coated vesicle (H+)-ATPase functions to provide the acidic environment within endosomes and other intracellular compartments necessary for receptor recycling and intracellular membrane traffic. The coated vesicle (H+)-ATPase is composed of nine different subunits which are divided into two distinct domains. The peripheral V1 domain, which has the structure 73(3):58(3):40(1):34(1):33(1), possesses the nucleotide binding sites of the (H+)-ATPase. The integral V0 domain, which has the composition 100(1):38(1):19(1):17(6), contains the pathway for proton conduction across the membrane. Topographical analysis indicates a structure for the coated vesicle (H+)-ATPase very similar to that of the F-type ATPases. Reassembly studies have allowed us to probe the function of particular subunits in this complex and the activity properties of the separate domains. These studies have led to insights into possible mechanisms of regulating vacuolar acidification.


The structure and biochemistry of the vacuolar H+ ATPase in proximal and distal urinary acidification. Vacuolar H+ ATPases participate in renal hydrogen ion secretion in both the proximal and distal nephron. These plasma membrane forms of the vacuolar H+ ATPase are regulated physiologically to maintain the acid-base balance of the organism. Proton transporting renal cells have requirements for constitutive acidification of intracellular compartments for normal endocytic and secretory functions. Recent experiments have begun to reveal how the kidney regulates these proton pumps independently. Vacuolar H+ ATPases are a family of structurally similar enzyme which differ in the composition of specific subunits. Cytosolic regulatory enzymes are present in renal cells which may affect vacuolar H+ ATPases in certain membrane compartments selectively. The vacuolar H+ ATPase in the plasma membrane of intercalated cells resides in a specialized proton-transporting apparatus that translocates the enzyme between an intracellular membrane pool and the plasma membrane in response to physiologic stimuli.


The vacuolar ATPase of Neurospora crassa. The filamentous fungus Neurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8-10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V1 and an integral membrane complex called V0. Genes encoding three of the subunits have been isolated. vma-1 and vma-2 encode polypeptides homologous to the alpha and beta subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis. vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.


Vacuolar H(+)-translocating ATPases from plants: structure, function, and isoforms. The vacuolar H(+)-translocating ATPase (V-type ATPase) plays a central role in the growth and development of plant cells. In a mature cell, the vacuole is the largest intracellular compartment, occupying about 90% of the cell volume. The proton electrochemical gradient (acid inside) formed by the vacuolar ATPase provides the primary driving force for the transport of numerous ions and metabolites against their electrochemical gradients. The uptake and release of solutes across the vacuolar membrane is fundamental to many cellular processes, such as osmoregulation, signal transduction, and metabolic regulation. Vacuolar ATPases may also reside on endomembranes, such as Golgi and coated vesicles, and thus may participate in intracellular membrane traffic, sorting, and secretion. Plant vacuolar ATPases are large complexes (400-650 kDa) composed of 7-10 different subunits. The peripheral sector of 5-6 subunits includes the nucleotide-binding catalytic and regulatory subunits of approximately 70 and approximately 60 kDa, respectively. Six copies of the 16-kDa proteolipid together with 1-3 other subunits make up the integral sector that forms the H+ conducting pathway. Isoforms of plant vacuolar ATPases are suggested by the variations in subunit composition observed among and within plant species, and by the presence of a small multigene family encoding the 16-kDa and 70-kDa subunits. Multiple genes may encode isoforms with specific properties required to serve the diverse functions of vacuoles and endomembrane compartments.


Subunit composition, biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase. The yeast vacuole is acidified by a vacuolar proton-translocating ATPase (H(+)-ATPase) that closely resembles the vacuolar H(+)-ATPases of other fungi, animals, and plants. The yeast enzyme is purified as a complex of eight subunits, which include both integral and peripheral membrane proteins. The genes for seven of these subunits have been cloned, and mutant strains lacking each of the subunits (vma mutants) have been constructed. Disruption of any of the subunit genes appears to abolish the function of the vacuolar H(+)-ATPase, supporting the subunit composition derived from biochemical studies. Genetic studies of vacuolar acidification have also revealed an additional set of gene products that are required for vacuolar H(+)-ATPase activity, but may not be part of the final enzyme complex. The biosynthesis, assembly, and targeting of the enzyme is being elucidated by biochemical and cell biological studies of the vma mutants. Initial results suggest that the peripheral and integral membrane subunits may be independently assembled.


Genetic and cell biological aspects of the yeast vacuolar H(+)-ATPase. The yeast vacuolar proton-translocating ATPase is a member of the third class of H(+)-pumping ATPase. A family of this type of H(+)-ATPase is now known to be ubiquitously distributed in eukaryotic vacuo-lysosomal organelles and archaebacteria. Nine VMA genes that are indispensable for expression of the enzyme activity have been cloned and characterized in the yeast Saccharomyces cerevisiae. This review summarizes currently available information on the VMA genes and cell biological functions of the VMA gene products.


Structural conservation and functional diversity of V-ATPases. The vacuolar system of eukaryotic cells contains a large number of organelles that are primary energized by an H(+)-ATPase that was named V-ATPase. The structure and function of V-ATPases from various sources was extensively studied in the last few years. Several genes encoding subunits of the enzyme were cloned and sequenced. The sequence information revealed the relations between V-ATPases and F-ATPases that evolved from common ancestral genes. The two families of proton pumps share structural and functional similarity. They contain distinct peripheral catalytic sectors and hydrophobic membrane sectors. Genes encoding subunits of V-ATPase in yeast cells were interrupted to yield mutants that are devoid of the enzyme and are sensitive to pH and calcium concentrations in the medium. The mutants were used to study structure, function, molecular biology, and biogenesis of the V-ATPase. They also shed light on the functional assembly of the enzyme in the vacuolar system.


Evolution of structure and function of V-ATPases. Proton pumping ATPases/ATPsynthases are found in all groups of present-day organisms. The structure of V- and F-type ATPases/ATP synthases is very conserved throughout evolution. Sequence analysis shows that the V- and F-type ATPases evolved from the same enzyme already present in the last common ancestor of all known extant life forms. The catalytic and noncatalytic subunits found in the dissociable head groups of the V/F-type ATPases are paralogous subunits, i.e., these two types of subunits evolved from a common ancestral gene. The gene duplication giving rise to these two genes (i.e., encoding the catalytic and noncatalytic subunits) predates the time of the last common ancestor. Mapping of gene duplication events that occurred in the evolution of the proteolipid, the noncatalytic and the catalytic subunits, onto the tree of life leads to a prediction for the likely subunit structure of the encoded ATPases. A correlation between structure and function of V/F-ATPases has been established for present-day organisms. Implications resulting from this correlation for the bioenergetics operative in proto-eukaryotes and in the last common ancestor are presented. The similarities of the V/F-ATPase subunits to an ATPase-like protein that was implicated to play a role in flagellar assembly are evaluated. Different V-ATPase isoforms have been detected in some higher eukaryotes. These data are analyzed with respect to the possible function of the different isoforms (tissue specific, organelle specific) and with respect to the point in their evolution when these gene duplications giving rise to the isoforms had occurred, i.e., how far these isoforms are distributed.


Absence of G(i) proteins in the Sf9 insect cell. Characterization of the uncoupled recombinant N-formyl peptide receptor. We investigated the interaction of the N-formyl peptide receptor (NFPR) with G proteins in infected Sf9 insect cells expressing the recombinant NFPR. Recombinant receptor expression of up to 27 pmol/mg protein was achieved in these cells. The receptor was recognized by an antiserum raised against an NFPR carboxyl-terminal peptide, and displayed specific and saturable binding of the formyl peptide ligand fMet-Leu-[3H]Phe. Scatchard analysis of the binding data yielded a dissociation constant of approximately 62 nM, a binding affinity of 60- to 120-fold lower than that of the high affinity sites in neutrophils and in transfected mammalian cell lines expressing the NFPR. That this low binding affinity was due to a lack of receptor coupling to G protein was suggested by the failure of guanine nucleotides to regulate receptor affinity and by the lack of formyl peptide-stimulated GTPase activity in these cells. Furthermore, immunoblotting with an anti-G(i) antibody and ADP-ribosylation experiments indicated that the approximately 40-kDa G(i) alpha subunit, which couples to the NFPR in neutrophils, is not present in Sf9 cell membranes. Thus, the current study provides for the first time evidence that a major G protein is absent in the Sf9 insect cells. Potential applications of the Sf9 system for in vitro reconstitution of the NFPR-G protein interaction are discussed.


A novel mechanism for regulation of vacuolar acidification. We have recently demonstrated that Cys-254 of the 73-kDa A subunit of the clathrin-coated vesicle (H+)-ATPase is responsible for sensitivity of the enzyme to sulfhydryl reagents (Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817-5822). In the present study we observe that for the purified enzyme, disulfide bond formation causes inactivation of proton transport which is reversed by dithiothreitol (DTT). DTT also restores activity of the oxidized enzyme following treatment with N-ethylmaleimide (NEM). These results indicate that disulfide bond formation between the NEM-reactive cysteine (Cys-254) and a closely proximal cysteine residue leads to inactivation of the (H+)-ATPase. To test whether sulfhydryl-disulfide bond interchange may play a role in regulating vacuolar acidification in vivo, we have determined what fraction of the (H+)-ATPase is disulfide-bonded in native clathrin-coated vesicles. Vesicles were isolated under conditions that prevent any change in the oxidation state of the sulfhydryl groups. NEM treatment of vesicles causes nearly complete loss of activity while subsequent treatment with DTT restores 50% of the activity of the fully reduced vesicles. By contrast, treatment of fully reduced vesicles with NEM leads to inactivation which is not reversed by DTT. These results indicate that a significant fraction of the clathrin-coated vesicle (H+)-ATPase exists in an inactive, disulfide-bonded state and suggest that sulfhydryl-disulfide bond interconversion may play a role in controlling vacuolar (H+)-ATPase (V-ATPase) activity in vivo.


Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii. To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.


Exon/intron structure of the human alpha 3(IV) gene encompassing the Goodpasture antigen (alpha 3(IV)NC1). Identification of a potentially antigenic region at the triple helix/NC1 domain junction. The Goodpasture antigen has been identified as the non-collagenous (NC1) domain of alpha 3(IV), a novel collagen IV chain (Saus, J., Wieslander, J., Langeveld, J., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the exon/intron structure and sequence for 285 amino acids of human alpha 3(IV), comprising 53 amino acids of the triple-helical domain and the complete NC1 domain (232 amino acids), were determined. Based on the comparison of the amino acid sequences of the alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) NC1 domains, a phylogenetic tree was constructed which indicates that alpha 2(IV) was the first chain to evolve, followed by alpha 3(IV), and then by alpha 1(IV) and alpha 5(IV). The exon/intron structure of these domains is consistent with this evolution model. In addition, it appears that alpha 3(IV) changed most after diverging from the parental gene. Analysis of its primary structure reveals that, at the junction between the triple-helical and NC1 domains, there exists a previously unrecognized, highly hydrophilic region (GLKGKRGDSGSPATWTTR) which is unique to the human alpha 3(IV) chain, containing a cell adhesion motif (RGD) as an integral part of a sequence (KRGDSGSP) conforming to a number of protein kinase recognition sites. Based on primary structure data, we outline new aspects to be explored concerning the molecular basis of collagen IV function and Goodpasture syndrome.


2,3,7,8-Tetrachlorodibenzo-p-dioxin causes reduction of glucose transporting activities in the plasma membranes of adipose tissue and pancreas from the guinea pig. Toxicity from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure results in severe metabolic imbalances leading to a loss of fat stores in many animal species, a phenomenon known as the wasting syndrome. In this paper, we report that TCDD treatment at very low doses (0.03-1 micrograms/kg, single intraperitoneal injection) causes a profound reduction of glucose uptake by guinea pig adipose tissue, pancreas and brain. This effect of TCDD is dose-dependent, with a dose as small as 0.03 micrograms/kg resulting in a significant decrease. In adipose tissue, the decrease begins within 6 h of treatment and persists at least 28 days. The Vmax of glucose transport was decreased by TCDD treatment, whereas the Km was unchanged. Liver behaves oppositely to adipose tissue. At early stages of treatment (6-12 h) glucose uptake was depressed, while at later stages (24-96 h) it was increased. In situ (explant tissue culture) treatment with TCDD yields similar trends as in vivo studies for glucose uptake in all three tissues. In adipose tissue culture TCDD starts reducing glucose uptake after 30 minutes. The inhibitory potencies of three dioxin congeners on adipose glucose transporting activities follows the same order of their toxicities in vivo. TCDD's effect on glucose transport is sensitive to cytochalasin B, a specific inhibitor of glucose transporter proteins. Based on these observations and the importance of glucose transporters to cellular energy maintenance, we conclude that at least in guinea pigs the reduction of glucose transporters in various tissues is one of the major causes for TCDD-induced wasting syndrome, which is so prominent in this species.


Enantiomeric selectivity of carbovir transport. Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay. The influx of both enantiomers appeared saturable and was inhibited greater than 90% by a combination of adenine (a low Km permeant of the nucleobase carrier) and dilazep (a potent inhibitor of nucleoside transport). The influx of (-)-CBV and (+)-CBV proceeded primarily via the nucleobase carrier with Vmax (picomoles/second/5 microliters of cells)/Km (millimolar) values of 17/0.12 and 140/1.9, respectively. To a lesser extent, the influx of (-)-CBV and (+)-CBV also occurred via the nucleoside transporter. Although both compounds exhibited a similar low affinity for this latter carrier (Km approximately 2 mM), the Vmax for (-)-CBV influx was approximately 4-fold higher than the Vmax for (+)-CBV influx. We conclude that both CBV enantiomers enter human erythrocytes by two transporters that are enantiomerically selective.


Identification of molecular aggregates containing glycoproteins III, J, K (carboxypeptidase H), and H (Kex2-related proteases) in the soluble and membrane fractions of adrenal medullary chromaffin granules. An investigation of the molecular properties of glycoprotein III has shown this to be a major component of molecular aggregates present in the membrane and soluble fractions of secretory vesicles from bovine adrenal medulla. These aggregates also contain components identified as glycoproteins H, J, and K which are molecular forms of Kex2-related proteases (glycoprotein H) and carboxypeptidase H (glycoprotein components J and K) and which have functions concerned with the processing of prohormones. A number of experiments indicated that these glycoproteins were associated. These components were coimmunoprecipitated from the soluble and membrane fractions of chromaffin granules. Purification of soluble glycoprotein III using wheat germ agglutinin-Sepharose resulted in the recovery of similar proportions of glycoproteins H, J, and K and gel filtration of the eluted material in combination with immunoprecipitation revealed the presence of heteroaggregates containing all of the glycoproteins. Similar results were obtained following octylglucoside solubilization of chromaffin granule membranes. Glycoprotein components III, H, J, and K were also found to have identical distributions following fractionation of chromaffin granule membranes with Triton X-114. It was concluded that the aggregates seen in the soluble fraction reflect an association of these components in the chromaffin granule membrane. This raises the possibility that these interactions are important for the targetting of these glycoproteins to secretory granules.


DNA melting within stable closed complexes at the Escherichia coli rrnB P1 promoter. Several transcription complexes are shown to form on the E. coli ribosomal rrnB P1 promoter in vitro. These include two closed complexes that are sensitive to heparin attack, and one open complex. The closed complexes are unusual in that they are both highly specific and stable, properties associated with the atypical DNA sequence of this promoter. The effector ppGpp does not prevent closed complex formation but does reduce the level of open complexes that form.


Tissue-specific DNaseI hypersensitivity regions are located in the 5'-region of the rat preproenkephalin gene. Extracellular stimuli affecting mechanisms of transcriptional control of the preproenkephalin gene link stimulus-secretion-synthesis coupling to transmitter phenotypic expression. In order to define distal and proximal genomic regions that might participate in transcriptionally active protein-DNA interactions, DNaseI hypersensitivity assays were conducted. Four DNaseI-hypersensitive regions were identified at -3000, -2500, -1800, and -200 base pairs (bp) relative to the somatic start site. The appearance of a site over the proximal promoter/enhancer region (-200 bp) is of interest since we and others have published a detailed footprint and functional analysis of this region using rat tissues. While liver (which does not express proenkephalin mRNA) displays essentially no hypersensitivity here, adrenal gland preparations show a strong signal and striatal tissue a significantly weaker signal. Cholinergic induction of proenkephalin mRNA was not associated with a change in hypersensitivity pattern. These data suggest that chromatin structure may have an influence on preproenkephalin transcriptional control and that cholinergic-inducible cis-acting DNA elements may reside within subdomains of at least these four hypersensitivity sites. Understanding the basis of this open chromatin structure should aid in identifying genomic mechanisms involved in tissue-specific expression and suggest avenues of future study.


Role of cadmium in activating nuclear protein kinase C and the enzyme binding to nuclear protein. Specific effects of cadmium on nuclear protein kinase C activity were found with 3T3/10T1/2 mouse fibroblast and rat liver nuclei. Treatment of the mouse fibroblasts in culture with 12-O-tetradecanoylphorbol-13-acetate resulted in the stimulation of nuclear protein kinase C activity in a "fixed" pool which is defined by its resistance to chelator extraction, whereas the chelator extractable enzyme activity, defined as the "labile" pool was unaffected. Cadmium was found to potentiate the effect of the phorbol ester, directed specifically to nuclei, since the particulate protein kinase C activity was not changed under similar treatment. In a reconstituted system consisting of rat liver nuclei and rat brain protein kinase C, cadmium stimulated the binding of the enzyme to a 105-kDa nuclear protein. The binding of a 105-kDa protein to protein kinase C is attributed strictly due to the cadmium effect, whereas a 50-kDa protein binding to protein kinase C was only enhanced by cadmium. We propose a mechanistic model, where cadmium substitutes zinc in the regulatory domain of protein kinase C rendering the putative protein-protein binding site exposed.


Molecular cloning of PEC-60 and expression of its mRNA and peptide in the gastrointestinal tract and immune system. The peptide PEC-60, structurally related to the pancreatic secretory trypsin inhibitor, inhibits glucose-induced insulin secretion. Here we report on the structure of a cDNA clone from pig duodenum encoding PEC-60. The cDNA encodes a 86-amino acid long precursor protein containing a 26-amino acid signal sequence, implying that the mature PEC-60 peptide is secreted from cells. Analysis of porcine duodenum demonstrated a high expression of a 0.6-kilobase long PEC-60 mRNA in this tissue, as well as the presence of strong PEC-60-like immunoreactivity in the cytoplasm of the majority of the goblet cells of the epithelium. High levels of PEC-60 mRNA were also found in the bone marrow and the peripheral blood and moderate levels in the spleen. A strong PEC-60-like immunoreactivity was localized in the monocytes of peripheral blood. Radioimmunoassay revealed high levels of pig PEC-60-like immunoreactivity in pig plasma suggesting that the PEC-60 peptide is efficiently released from cells. These findings imply that the gastrointestinal peptide PEC-60 is formed, stored, and secreted from monocytes present within the bone marrow and in the peripheral blood, indicating a role of the PEC-60 peptide in the immune system in addition to its function as a gastrointestinal peptide.


Assembly and expression of an intrinsic factor IX activator complex on the surface of cultured human endothelial cells. Endothelial cells expose specific receptors for blood clotting factors and, upon perturbation, can initiate and propagate the reactions of the extrinsic pathway of blood coagulation leading to fibrin formation on the cell surface. The existence of an intrinsic mechanism of Factor IX activation on cultured human umbilical vein cells (HUVECs) was investigated by studies of the interaction between HUVECs and two proteins of the contact activation system, the cofactor high molecular weight kininogen (H-kininogen) and the zymogen Factor XI. In the presence of zinc ions (10-300 microM), 125I-labeled H-kininogen bound to HUVECs in a time-dependent, reversible, and saturable manner, with calcium ions exerting an inhibitory effect on the zinc-dependent binding. Analysis of the binding data by the LIGAND computer program indicated that HUVECs, in the presence of 2 mM CaCl2 and 100 microM ZnCl2 at 37 degrees C, bound 1.14 x 10(7) H-kininogen molecules per cell with an apparent dissociation constant of 55 nM. HUVEC-bound H-kininogen functions as the cell surface receptor for both 125I-labeled Factor XI and 125I-labeled Factor XIa, since HUVECs cultured in contact factor-depleted serum do not detectably bind either the zymogen or the enzyme in the absence of H-kininogen and zinc ions. In the presence of saturating concentrations of H-kininogen, 2 mM CaCl2 and 100 microM ZnCl2, the binding of 125I-labeled Factor XI and Factor XIa to HUVECs was time-dependent, reversible, and saturable, with apparent dissociation constants of 4.5 and 1.5 nM, respectively. HUVEC-bound complexes of H-kininogen and Factor XI generated Factor XIa activity only after the addition of purified Factor XIIa, and cell-bound Factor XIa in turn activated Factor IX, as documented by a 3H-labeled activation peptide release assay for 3H-Factor IX activation. The results indicate that cultured HUVECs provide a surface for the assembly and expression of an intrinsic Factor IX activator complex that may participate in the initiation of blood coagulation at sites of vascular injury.


Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells. Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.


Epoxyeicosatrienoic acids inhibit Ca2+ entry into platelets stimulated by thapsigargin and thrombin. The epoxyeicosatrienoic acids derived from the cytochrome P-450 pathway of arachidonic acid metabolism have a unique platelet antiaggregatory profile. This prompted us to examine their influence on cellular Ca2+ mobilization. 14,15-cis-Epoxyeicosatrienoic acid and related compounds inhibited the rise in cytosolic Ca2+ following agonist stimulation of platelets by thapsigargin, a receptor-independent agonist, and thrombin, a receptor-dependent agonist. The epoxyeicosatrienoic acids selectively inhibited the entry of Ca2+ from the exterior of the platelets but did not alter Ca2+ discharge from intracellular pools. The magnitude of inhibition by 14,15-cis-epoxyeicosatrienoic acid was proportional to the rate of Ca2+ entry. 14,15-cis-Epoxyeicosatrienoic acid also inhibited the rate of influx of Mn2+, a cation which enters platelets via pathways similar to Ca2+. The magnitude of inhibition was proportional to the rate of Mn2+ entry, suggesting that epoxyeicosatrienoic acids act on divalent cation channels in a fashion which depends on the state of opening of the channel. Selective inhibition of Ca2+ entry into platelets may account for the antiaggregatory effects of the epoxyeicosatrienoic acids. We are unaware of other endogenous compounds exhibiting this property, suggesting that epoxyeicosatrienoic acids may be useful to probe agonist-stimulated Ca2+ mobilization in nonexcitable cells.


Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis. The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.


Mechanism and stereochemistry in the sequential enzymatic saturation of the two double bonds in cholesta-4,6-dien-3-one. The mechanism and stereochemistry in connection with enzymatic conversion of cholesta-4,6-dien-3-one into cholestanol was studied. Rat and mouse liver microsomes are able to catalyze NADPH-dependent sequential saturation of the two double bonds. Evidence was obtained that the saturation of the delta 6-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 7-position of the steroid (mainly 7 beta-position), followed by addition of a proton to the 6 alpha-position (mainly trans addition). The saturation of the delta 4-double bond includes transfer of a hydride ion from the B-side of the cofactor to the 5 alpha-position of the steroid followed by addition of a proton to the 4 beta-position (trans addition). The reduction of the 3-oxo group was found to involve transfer of a hydride ion from the B-side of the cofactor NADPH to the 3 alpha-position of the steroid. The results are in accord with the contention that the enzymatic saturation of the two double bonds involves a polarization of the 3-oxo group making C-7 electrophilic and C-6 nucleophilic in connection with the saturation of the delta 6-double bond and C-5 electrophilic and C-4 nucleophilic in connection with the saturation of the delta 4-double bond.


Pulmonary surfactant secretion is regulated by the physical state of extracellular phosphatidylcholine. Pulmonary alveolar type II cells synthesize, secrete, and recycle the components of pulmonary surfactant. In this report we present evidence that dipalmitoylphosphatidylcholine is a potent inhibitor of surfactant lipid secretion by type II cells. Monoenoic and dienoic phosphatidylcholines with fatty acids of 16 or 18 carbons are ineffective as inhibitors of surfactant lipid secretion. In contrast, disaturated phosphatidylcholines, with either symmetric or asymmetric pairs of fatty acids of 14, 16, or 18 carbons, exhibit inhibition of surfactant secretion that correlates extremely well with the phase transition temperature (Tc) of the phospholipid. The inhibitory activity of dipalmitoylphosphatidylcholine is not dependent upon lipid stereochemistry. N-Methylated derivatives of dipalmitoylphosphatidylethanolamine are significantly less effective than phosphatidylcholine as inhibitors. Phosphatidylcholines below their phase transition temperature are inhibitors of surfactant secretion, whereas those above their phase transition temperature are either ineffective or weakly inhibitory. The phase transition dependence of inhibition is observed when type II cells are incubated at 37 degrees C with different species of phosphatidylcholine. In addition, if type II cells are stimulated to secrete at different temperatures the efficacy of a given phospholipid as an inhibitor is dependent on its relationship to Tc (i.e. dipalmitoylphosphatidylcholine with a Tc of 41 degrees C significantly inhibits secretion at 37 degrees C but not at 42 degrees C). Inhibition of surfactant secretion by dipalmitoylphosphatidylcholine is abrogated when it is incorporated into the same liposome with dioleoylphosphatidylcholine as a 50:50 mixture. In contrast, the simultaneous addition of two separate populations of liposomes, one composed of dipalmitoylphosphatidylcholine and the other composed of dioleoylphosphatidylcholine, does not significantly alter the inhibitory activity found with dipalmitoylphosphatidylcholine alone. These data provide compelling evidence that the physical state of phosphatidylcholine can regulate surfactant secretion from alveolar type II cells and suggest a unique mechanism for regulating exocytosis in the alveolus of the lung.


Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase. A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.


Characterization of the interaction between the heavy and light chains of bovine factor Va. Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.


Secondary structure and folding of a functional chloroplast precursor protein. Ferredoxin is a chloroplast stroma protein which is cytosolically synthesized as a precursor with an amino-terminal extension called the transit sequence that is needed for the post-translational uptake by the chloroplast. To characterize the secondary and tertiary structure elements, the full precursor, the holo- and apo- (without iron-sulfur cluster) forms of the mature protein, and the chemically synthesized transit peptide were obtained and analyzed separately. Circular dichroism, tryptophan fluorescence quenching, and protease accessibility experiments indicate that the precursor has a low content of defined secondary structure and resembles unfolded proteins; these properties are due to both the mature part and the transit sequence. This result provides an explanation for the lack of cytosolic factor requirement of this protein for import. In an import competition assay, the isolated transit peptide had an affinity for the chloroplasts comparable to the full precursor. Interestingly and of possible importance to the import process, the transit peptide has conformational flexibility as it adopts alternative secondary structures in different environments.


Increased exposure of hydrophobic surface in molten globule state of alpha-lactalbumin. Fluorescence and hydrophobic photolabeling studies. The involvement of molten globule state as a distinct intermediate in the denaturation process in proteins is well documented. However, the structural characterization of such an intermediate is far from complete. We have, using fluorescence and fluorescence quenching, studied the molten globule state of bovine alpha-lactalbumin. Unlike the native state, where all the 4 tryptophans are buried in the protein, 2 tryptophans are exposed in the molten globule state. Using the hydrophobic photoactivable reagent [3H]diazofluorene, we observe an increased hydrophobic exposure in the molten globule state. These structural characteristics conform to the current views on the molten globule state, i.e. it has similar secondary structure but a poorly defined tertiary structure. Our fluorescence studies indicate the involvement of a premolten globule state in the native to molten globule state transition. This premolten globule state exists at pH 5.0 and has a very compact structure involving increased hydrophobic interactions in the protein interior. These results are also supported by circular dichroism studies.


Mucin biosynthesis revisited. The enzymatic transfer of Gal in beta 1,3 linkage to the GalNAc moiety of the core structure R1-GlcNAc beta 1,6GalNAc alpha-O-R2. Synthetic glycosides containing the core, -Glc-NAc beta 1,6GalNAc alpha-, acted as acceptors for beta-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the alpha-benzylglycoside of LacNAc beta 1,6GalNAc (LGBn) (25.1 nmol of Gal) and the alpha-ortho-nitrophenylglycosides of LacNAc beta 1,6GalNAc (22.0 nmol of Gal), GlcNAc beta 1,6GalNAc (15.5 nmol of Gal), and Fuc alpha 1,3GlcNAc beta 1,6GalNAc (25.9 nmol of Gal); LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the alpha-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. The alpha GalNAc:beta 1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum----1.4; ascitic fluid----0.9; tumor----17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37 degrees C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37 degrees C was continued for 40 h at 4 degrees C, indicating the high affinity of LGBn for the enzyme at lower temp. Km for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; Km for ACGM (M(r) 200,000) in presence of LGBn was 16.1 microM and Ki for ACGM (as the inhibitor) was 41.7 microM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fuc alpha 1,3GlcNAc beta 1,6GalNAc alpha-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of beta 1,3Gal attachment on alpha GalNAc, which has already been substituted by beta 1,6GlcNAc, then elongated by beta 1,4Gal and also terminated by alpha 1,3Fuc.


Identification of an activating transcription factor (ATF) binding site in the human transforming growth factor-beta 2 promoter. Transforming growth factor TGF-beta 2 is encoded by multiple mRNA transcripts of 5.8, 5.1, 4.0, 3.8, and 2.8 kilobase pairs (kb) that are expressed in various human and monkey cells. Northern blot analysis using genomic fragments of DNA was used to demonstrate that some of this size heterogeneity is due to differences in the length of the 5'-untranslated region. Probes that were colinear with the first 600 nucleotides of the 5'-untranslated region detected only the 5.8-, 4.0-, and 3.8-kb transcripts. In order to identify DNA elements that regulate the transcription of these mRNA transcripts, deletion constructs of 5'-flanking DNA were ligated to the coding region for chloramphenicol acetyltransferase (CAT) and analyzed for promoter activity in several cell lines. Sequences responsible for putative enhancer and silencer regions were identified between -778 and -40 relative to the transcription initiation site. Addition of a cyclic AMP-responsive element/activating transcription factor-like element at -74 resulted in a 5-10-fold increase in CAT activity over that expressed with a construct that contained only the TATA box. This increase in CAT activity was suppressed by the addition of DNA sequences between -257 and -187, whereas sequences between -778 and -257 stimulated CAT activity. Point mutations within the ATF binding site at -74 resulted in a marked decrease in CAT expression. Cotransfection with ATF-1 or ATF-2 expression plasmids resulted in both dose-dependent stimulatory and inhibitory activities that were cell type-dependent. These studies identify multiple transcription initiation sites for TGF-beta 2 and demonstrate that transcription from one of these promoters is dependent upon an ATF binding site located 5' of the TATA box.


The complete amino acid sequence of yam (Dioscorea japonica) chitinase. A newly identified acidic class I chitinase. The complete amino acid sequence of acidic chitinase from yam (Dioscorea japonica) aerial tubers was determined. The protein is composed of a single polypeptide chain of 250 amino acid residues and has a calculated molecular mass of 27,890 Da. There is an NH2-terminal domain, a hinge region, and a main structure, typical for class I chitinases (Shinshi, H., Neuhaus, J.-M., Ryals, J., and Meins, F., Jr. (1990) Plant Mol. Biol. 14, 357-368). We have obtained the first evidence for an acidic class I chitinase. Comparison with sequences of other class I chitinases revealed approximately 40% sequence similarity, a value lower than that for other class I chitinases (70-80%). We assume that there is a local conformational change in the molecule; cysteine residues that probably form disulfide bonds are completely conserved, with the exception of Cys-178. The difference in structure between this chitinase and other basic class I chitinases suggests that acidic and basic isoforms should be grouped into subclasses; this protein is an ethylene- or a pathogen-independent chitinase produced by a gene that is inherent in the tuber.


Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides. A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.


Inhibition of mitogen-induced c-fos expression in melanoma cells by retinoic acid involves the serum response element. To investigate the mechanism(s) by which all-transretinoic acid (RA) inhibits cell growth, we studied its effect on the expression of c-fos and c-jun in B16 melanoma cells. RA differentially inhibited proto-oncogene induction by mitogens, such as phorbol 12-myristate 13-acetate and serum. Suppression of c-fos was achieved with doses of RA as low as 10(-10) M and required pretreatment of cells with RA for a minimum of 2 h. In contrast, inhibition of c-jun required pretreatment for greater than 16 h with at least 10(-8) M RA and coincided with the observed decrease in cell growth. RA blocked c-fos induction by inhibiting transcription. This inhibition of transcription occurs through the serum response element (SRE), since the SRE alone was sufficient to confer down-regulation by RA to a minimal c-fos promoter construct. Thus, the SRE plays a critical role in the suppression of c-fos transcription by RA.


Characterization of the tryptophanase operon of Proteus vulgaris. Cloning, nucleotide sequence, amino acid homology, and in vitro synthesis of the leader peptide and regulatory analysis. The tryptophanase (tna) operon of Proteus vulgaris was cloned and characterized and found to be organized similarly to the tna operon of Escherichia coli. Both operons contain two major structural genes, tnaA and tnaB, that encode tryptophanase and a tryptophan permease, respectively. tnaA of P. vulgaris is preceded by a transcribed leader region, encoding a 34-residue leader peptide, TnaC, that contains a single tryptophan residue. The tnaC coding region also has a boxA-like sequence. Regulatory studies performed in P. vulgaris, and with a plasmid carrying the P. vulgaris tna operon in E. coli, established that expression of the Proteus operon was induced by tryptophan and was subject to catabolite repression. Site-directed mutagenesis studies established that translation of the tnaC coding region was essential for induction. Synthesis of the P. vulgaris leader peptide was demonstrated in an in vitro coupled transcription-translation system. Interestingly, the 5 amino acid residues of the TnaC peptide surrounding the sole tryptophan residue are identical in P. vulgaris and E. coli. We conclude that the tna operon of P. vulgaris is also regulated by tryptophan-induced transcription antitermination. Homology of tryptophanase and tryptophan permease of P. vulgaris to related proteins from other species is described.


A novel CoA-independent transacetylase produces the ethanolamine plasmalogen and acyl analogs of platelet-activating factor (PAF) with PAF as the acetate donor in HL-60 cells. In this study, we demonstrate the presence of a unique membrane-associated transacetylase that transfers the acetate group from platelet-activating factor (PAF) to lysoplasmalogen (in the presence of EDTA and sodium acetate) with the formation of 1-alk-1-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (alk-1-enylacetyl-GPE). The identity of alk-1-enylacetyl-GPE was confirmed by acid hydrolysis, phospholipases A2 or C treatment and derivatization by fluorodinitrobenzene. The transacetylase has no requirement for Ca2+, Mg2+, or CoA and a broad pH optimum (7.0-8.0) with Km values of 12.0 microM for PAF and 106.4 microM for lysoplasmalogens. The enzyme activity from the isolated membrane fraction is not changed when whole cells are supplemented with 20:4, induced to differentiate into granulocytes, or treated with ionophore A23187. Radyllyso-sn-glycero-3-phosphocholine (GPC), radyllyso-GPE, acyllyso-sn-glycero-3-phosphoserine (GPS), acyllyso-sn-glycero-3-phosphoinositol (GPI), alkyllyso-sn-glycero-3-phosphate (GP), acyllyso-GP, or cis-9-octadecen-1-ol can also serve as acetate acceptors, whereas alkylglycerol, acylglycerol, or cholesterol are inactive. Differences in substrate acceptor specificity, sensitivity toward phenylmethylsulfonyl fluoride, and response to temperature suggest that the CoA-independent transacetylase and the CoA-independent transacylase that transfers long-chain acyl moieties are two separate enzymes. With intact differentiated HL-60 cells, [3H]acetate from [3H]PAF can be incorporated into alk-1-enylacetyl-GPE in the presence of ionophore A23187, but not in its absence. Moreover, phospholipase A2 inhibitors (p-bromophenacyl bromide and mepacrine) block the transacetylation process in whole cell system. These results indicate the production of alk-1-enyllyso-GPE is a rate-limiting factor for the subsequent transacetylation step during cell activation. We conclude that the transacetylase may participate in the biosynthesis of ethanolamine plasmalogen and acyl analogs of PAF, in vivo, fine-tuning of PAF biological responses, and cross-talk between de novo and remodeling pathways of PAF biosynthesis.


Expression of mature bovine H-protein of the glycine cleavage system in Escherichia coli and in vitro lipoylation of the apoform. H-protein, a component of the glycine cleavage system with lipoic acid as a prosthetic group, was expressed in Escherichia coli using a T7 RNA polymerase plasmid expression system. After induction with 25 microM isopropyl-beta-D-thiogalactopyranoside, bacteria harboring the recombinant plasmid expressed mature bovine H-protein as a soluble form at a level of about 10% of the total bacterial protein. Little of the H-protein was lipoylated in E. coli cultured without added lipoate, but when the cells were cultured in medium supplemented with 30 microM lipoate, about 10% of the recombinant protein expressed was the correctly lipoylated active form, 10% was an inactive aberrantly modified form, presumably with an octanoyl group, and the remaining 80% was the unlipoylated apoform. Each of the three forms was purified to homogeneity and shown to have the same NH2-terminal amino acid sequence as that of native bovine H-protein. The specific activity of the lipoylated form of H-protein expressed was consistent with that of H-protein purified from bovine liver. The purified recombinant apo-H-protein was lipoylated and consequently activated in vitro with lipoyl-AMP as a lipoyl donor by lipoyltransferase purified 150-fold from bovine liver mitochondria. The lipoylation was dependent on lipoyl-AMP, apo-H-protein, and lipoyltransferase. The partially purified lipoyltransferase had no lipoate-activating activity. These results provide the first evidence that in mammals two consecutive reactions are required for the attachment of lipoic acid to the acceptor protein: the activation of lipoic acid to lipoyl-AMP catalyzed by lipoate-activating enzyme and the transfer of the lipoyl group to an N epsilon-amino group of a lysine residue to apoprotein by lipoyl-AMP:N epsilon-lysine lipoyltransferase.