Subcutaneous fat accumulation during pregnancy in a malnourished population. 1. Total skinfold thicknesses (sum of seven sites) were measured in fifty-seven pregnant women with low energy and protein intake, at weeks 24, 30 and 35 of gestation. 2. Women were classified in two groups (overweight and low weight), according to their weight-for-height at week 24 of gestation. 3. Half of the women did not increase, or even reduced the amount of subcutaneous fat during the observation period. The lowest mean value of total skinfold thickness was found in the low-weight women who showed a reduction of the subcutaneous fat. 4. This finding shows that a significant proportion of malnourished women do not follow the average pattern of subcutaneous fat accumulation seen in healthy pregnant women without food intake restriction, during the second half of pregnancy.


Studies on the absorption of calcium, phosphorus, magnesium, copper and zinc by sheep fed on roughage-cereal diets. 1. Two groups of four 18-month-old ewes fitted with a rumen cannula and re-entrant cannulas in the proximal duodenum were fed on one of two diets based on varying proportions of dried-grass meal, ground straw and barley. 2. The apparent availability, retention and amount of the mineral reaching the proximal duodenum were measured for calcium, phosphorus, magnesium, copper and zinc. Significant differences were found between diets for Ca only. Net absorption of Mg occurred before the proximal duodenum and also, lower down the alimentary tract. Marked increases were observed in the amount of Cu, Zn and P reaching the proximal duodenum relative to that ingested.


Generalized equations for predicting body density of men. 1. Skinfold thickness, body circumferences and body density were measured in samples of 308 and ninety-five adult men ranging in age from 18 to 61 years. 2. Using the sample of 308 men, multiple regression equations were calculated to estimate body density using either the quadratic or log form of the sum of skinfolds, in combination with age, waist and forearm circumference. 3. The multiple correlations for the equations exceeded 0.90 with standard errors of approximately +/- 0.0073 g/ml. 4. The regression equations were cross validated on the second sample of ninety-five men. The correlations between predicted and laboratory-determined body density exceeded 0.90 with standard errors of approximately 0.0077 g/ml. 5. The regression equations were shown to be valid for adult men varying in age and fatness.


The effect of dietary protein intake on calcium metabolism of the pregnant ewe. 1. The effect of protein intake on calcium metabolism has been studied in the pregnant ewe. 2. Results suggest that decreased Ca retention in protein-deficient animals was a result of a decreased rate of Ca absorption and not a decreased rate of bone accretion as suggested by other workers.


Absorption of inorganic ions and volatile fatty acids in the rabbit caecum. 1. Rabbit caecal segments in situ were used to measure absorption rates of volatile fatty acids (VFA) and inorganic ions from a saline solution comparable in composition to normal caecal fluid. 2. Results confirm the importance of VFA absorption from caecal material found by other workers. 3. Like the mammalian colon, the rabbit caecum conserved large amounts of sodium, chloride and water. Bicarbonate was also absorbed. 4. VFA replacement studies showed that net water absorption was reduced, net electrolyte absorption was hardly influenced. 5. Na replacement completely inhibited net water absorption and decreased net VFA and Cl absorption, HCO3 was heavily secreted. 6. These findings indicate that VFA absorption in the rabbit caecum is partly dependent on Na absorption and that in the absence of Na an anion-exchange mechanism occurs.


Effect of vitamin B12 deficiency on phosphatidylethanolamine methylation in rat liver. 1. In vitamin B12 deficiency the activity of tetrahydropteroylglutamate methyltransferase (EC 2.1.1.13) is depressed and the synthesis of methionine is reduced. Because the methyl group of methionine is largely utilized for the methylation of phosphatidylethanolamine, we investigated the effects of vitamin B12 deficiency on phosphatidylcholine synthesis. 2. The incorporation of injected [14C]formaldehyde into liver phosphatidylcholine was reduced by approximately 50% in vitamin B12-deficient rats. Also the corresponding incorporation of 5-[14C]methyltetrahydrofolic acid tended to decrease. The findings are consistent with a lower conversion of these precursors to methionine. 3. The effect of the deficient methyl-group supply on phosphatidylcholine synthesis was also investigated by the injection of [14C]ethanolamine. The amount (%) of lipid-14C recovered in phosphatidylcholine was significantly reduced in vitamin B12 deficiency. 4. Chemical analysis of liver phospholipids showed that the vitamin B12-deficient rats had a higher proportion of phosphatidylethanolamine and a lower proportion of phosphatidylcholine, indicating that the impaired synthesis of phosphatidylcholine by methylation leads to changes in membrane phospholipid composition.


The effects of folic acid supplementation during pregnancy in the rat. 1. Twenty-four Sprague-Dawley female rats were randomly assigned to three groups (groups A, B, C). Group A was given a folic acid-free diet and groups B and C received 0.0018 g folic acid/kg diet. Rats in group C were also given a supplement of 1 mg folic acid/d by intraperitoneal injection. 2. After 14 d of feeding the rats were mated. The diets were continued throughout gestation. On day 21, of gestation the dams were killed and their livers and products of conception assayed for RNA, DNA, protein and tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate dehydrogenase; EC 1.5.1.3) activity. 3. The foetuses, placentas and livers from supplemented rats (group C) were significantly larger than those from groups A and B and had a higher content of RNA, DNA and protein. Those tissues from group A dams were smaller than those from the other groups and had a correspondingly reduced nucleic acid and protein content. 4. The activity of tetrahydrofolate dehydrogenase, the first and rate-limiting enzyme in the metabolism of folate, was increased in the folate supplemented rats (group C) and reduced in the rats given a folic acid-free diet (group A). These changes in enzyme activity could explain the differences in nucleic acid biosynthesis and growth shown by the different groups.


The effect of trenbolone acetate with time on the various responses of protein synthesis of the rat. 1. The anabolic agent trenbolone acetate (3-oxo-17beta-hydroxy-4,9,11-oestratriene acetate; TBA) given subcutaneously to female rats increased their growth rate compared with that of the placebo-treated controls. 2. The increased growth rate of the TBA-treated rats was not a consequence of an increased water retention. The TBA-treated rats had a significantly higher (P less than 0.01) total carcass nitrogen content but the total carcass fat content decreased by a non-significant (P greater than 0.05) 8.3%. 3. There was evidence of a time-lag in the response of the fractional synthetic rate of certain individual tissues to TBA. The fractional synthetic rates of the uterus and skeletal muscle mixed tissue proteins were significantly reduced in the TBA-treated rats. 4. The measured reduction in fractional synthetic rates was concluded to reflect true changes in the synthetic rate rather than a result of an alteration in the specific activity of the tyrosine pool used for protein synthesis.


The effects of fasting in Ramadan. 1. Serum uric acid and lipid concentrations. 1. The changes in serum levels of uric acid and lipids during 1 month of starvation-refeeding were measured in sixteen male volunteers. 2. Uric acid levels increased linearly with the duration of the experiment. The increase was positively correlated with the increase in serum triglycerides but not with cholesterol or phospholipids. 3. Triglycerides increased at a faster rate than uric acid implying that the increase in uric acid was secondary to that of the lipid. 4. It was concluded that the purine and lipid synthetic pathways are linked through a common small-molecular-weight effector rather than through the sharing of a common enzyme.


Kinetics of substrate and product interactions with arsanilazotyrosine-248 carboxypeptidase A. The chromophoric intramolecular azoTyr-248.Zn complex detects discrete kinetic steps in the interaction of azocarboxypeptidase with products or substrates that are hydrolyzed slowly. Temperature-jump experiments at 510 nm indicate that the rapid binding of such ligands is followed by a slower change in the conformation of the enzyme--ligand complex: that defines the initial binding, and the rate constants k2 and k-2 for the forward and reverse steps of this conversion, respectively. For each ligand, the kinetically determined dissociation constant is virtually identical to that obtained at equilibrium form circular dichroic titrations. Although there are small variations in k2 and k-2 for each substrate, all the rate processes are much faster than the rate-determining step for the hydrolysis of these substrates. The proposed model of the mechanism of peptide hydrolysis by carboxypeptidase incorporates the results of these temperature jump experiments.


Comparison of the 3alpha-and 20beta-hydroxysteroid dehydrogenase activities of the cortisone reductase of Streptomyces hydrogenans. The 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSD) activities of cortisone reductase in Streptomyces hydrogenans have been examined to determine whether both activities are due to one enzyme. This question was raised when changes in the commercial preparations of the enzyme reduced the 3alpha-HSD activity to 5% of its original level while retaining full 20beta-HSD activity. In our experiments, the enzyme was purified to crystallinity and partially characterized. The 3alpha- and 20beta-HSD activities were both coinduced and copurified. The 3alpha- and 20beta-HSD activities were compared using the crystalline enzyme for studies of substrate competition, thermal inactivation at 52 degrees C, loss of activity with three haloacetoxysteroids, and the effects of Me2SO and temperature on the reaction rate. These studies support the conclusion that the 3alpha- and 20beta-HSD activities are due to the same enzyme molecule. In addition, it appears that the binding sites for the two activities do not act independently.


Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments. Papain and thermolysin are shown to cleave bovine rhodopsin in native membranes in two temporally distinct steps at room temperature. The final product of the proteolysis consists of two membrane-bound fragments of molecular weights 27 000 (Rh27) and 12 500 (Rh12). The molecular weights are not changed by reduction with dithiothreitol. The two fragments remain closely associated in both the membrane and nondenaturing detergents before and after bleaching and can be selectively cross-linked with carbodiimides. The sulfhydryl chemistry of the cleaved protein in nearly indistinguishable from native rhodopsin, and of the total of six sulfhydryl groups, two are located on Rh12 and four on Rh27. In the membrane-bound protein, two sulfhydryl groups are accessible for modification, one on Rh12 and the other on Rh27. The sulfhydryl on Rh12 is particularly reactive and may be selectively labeled with maleimides. Continuous irradiation with white light induces additional sulfhydryl reactivity on Rh27.


Organization of rhodopsin in photoreceptor membranes. 2. Transmembrane organization of bovine rhodopsin: evidence from proteolysis and lactoperoxidase-catalyzed iodination of native and reconstituted membranes. Proteolysis of reconstituted membranes with papain and thermolysin reveals the existence of two rhodopsin populations: one susceptible to proteolysis and the other protected. The susceptible population corresponds to rhodopsin molecules with the same orientation as rhodopsin in the native membrane, while the protected population corresponds to "inverted" rhodopsin molecules only found in reconstituted membranes. Using an iodination enhancement probe, we demonstrate that lactoperoxidase catalyzes iodination of rhodopsin exclusively on the external surface of these sealed reconstituted vesicles. Furthermore, we find that both rhodopsin populations in reconstituted membranes (normal and inverted) are readily iodinated by lactoperoxidase, providing definitive evidence that the rhodopsin polypeptide spans the membrane thickness. Additional conclusions from these experiments are discussed in terms of a model for the folding of the rhodopsin polypeptide in the membrane.


Saccharide binding to transition metal ion free concanavalin A. Saccharide binding has been observed with demetallized concanavalin A in the presence of Ca(2+) only, using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside. At pH 7.2 both the nicked and intact forms of concanavalin A bound 4-methylumbelliferyl alpha-D-mannopyranoside with similar affinities. Competitive binding with methyl alpha-D-mannopyranoside was demonstrated. The association constants at 5 degrees C were 9.6 +/- 0.6 X 10(4) M(-1) for 4-methylumbelliferyl alpha-D-mannopyranoside and 1.1 +/- 0.3 X 10(4) M(-1) for methyl alpha-D-mannopyranoside. 4-Methylumbelliferyl alpha-D-mannopyranoside binding was also observed if demetallized concanavalin A was remetallized with less than stoichiometric amounts of Ca(2+). The association constants with low Ca(2+) concentrations were similar to those determined with saturating Ca(2+). With less than stoichiometric levels of Ca(2+), the number of sugar molecules bound per protein subunit was a reflection of the fraction of activated lectin subunits. These results show that saccharide binding activity of concanavalin A does not require a transition metal ion at pH 7.2; only Ca(2+) is required. At pH values near 5, where most previous studies have been carried out, both a transition metal ion and Ca(2+) are necessary.


Metal ion induced conformational transitions of prothrombin and prothrombin fragment 1. Circular dichroism experiments indicate that prothrombin fragment 1 undergoes essentially the same secondary structural change whether in the presence of Ca(2+), Mg(2+), or Mn(2+). Titration with any of these metal ions results in a sigmoidal titration curve indicative of cooperative binding. Mg(2+) and Ca(2+) have nearly identical transition midpoints, while that for Mn(2+) is an order of magnitude less. These results correlate well with the results of previous metal ion intrinsic fluorescence quenching experiments. Fragment 1 has previously been shown to undergo a second transition corresponding to dimerization at high calcium concentrations. The present circular dichroism experiments show that this transition does not result in a gross alteration of secondary structure in the fragment 1 molecule. Studies with prothrombin, similar to those with fragment 1, indicate a similar metal ion dependent conformational change but of smaller magnitude. As apparently only the fragment 1 portion of the molecule undergoes the transition, it would appear that the covalently linked fragment 1 is constrained from attaining the same conformation as the purified entity. This suggests that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin.


Conformational studies on [Pro3, Gly4]-oxytocin in dimethyl sulfoxide by 1H nuclear magnetic resonance spectroscopy: evidence for a type II beta turn in the cyclic moiety. A model for oxytocin has been previously proposed in which residues 3 and 4 occupy the corner positions in a beta turn (Urry, D. W., & Walter, R (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 956). The analogue [Pro(3),Gly(4)]-oxytocin (PGO) was used in proton magnetic resonance (1H NMR) studies designed to probe the contribution of these corner positions in forming a beta turn. Comparison of various 1H NMR parameters obtained at 220 MHz for backbone amide protons of PGO with those for the corresponding protons of oxytocin suggests that, to a first approximation, these two peptides may have similar backbone conformations in )CD3)2SO. Theoretically, the L-Pro lead toGly sequence in positions 3 and 4 of PGO would allow the formation of either a type I or type II beta turn. The two coupling constants between vicinal amide and C alpha protons for Gly(4) of PGO in (CD3)2SO are compatible with a type II beta turn in which 04, the dihedral angle about the bond between the backbone C alpha and N' atoms of Gly4, is appromximately +92 degrees, but not with a type I beta turn. A survey of peptides studied in other laboratories by X-ray and/or 1H NMR with reported type II beta turns with L-Pro lead toGly or Gly lead to Gly sequences in the corner positions revealed a close correlation between chemical shifts and vicinal coupling constants for the glycl residue in the second corner position. It is suggested that this criterion can form an additional basis for the characterization of beta turns. More studies are needed to determine the particular type of beta turn manifest in the cyclic moiety of oxytocin per se, although a reasonable working hypothesis is that oxytocin, similar to PGO, has a type II beta turn.


Transient electric dichroism studies of nucleosomal particles. We report transient electric  dichroism experiments on nucleosomal core particles containing 140 and 175 base pairs of DNA, and on spacerless dinucleosomes. The results indicate that all particles posses a permanent dipole moment. The orientation time of 140 base pair nucleosomes implies an estimated maximum dimension of a = 130 A (a must be at least 111 A), consistent with the disk model. The maximum dimension of the spacerless dinucleosome is estimated to be about 290 A (at least 180 A), ruling out a structure in which two disks are stacked directly on top of each other. The reduced dichroism amplitude indicate that the DNA superhelix axis in nucleosomes aligns perpendicular to the electric field, as expected for a dipole moment directed along a C2 symmetry axis across the disk diameter. Nucleosomes containing 175 base pairs of DNA show a substantially larger dichroism amplitude that do 140 base pair nucleosomes. In the context of the disk model, this result is shown to be consistent with 100 base pairs of DNA per superhelical turn, but not with 80 base pairs per turn.


Physical studies of nucleosome assemble. Biochemical, spectroscopic, and hydrodynamic studies were performed on the reconstituted complex of 140 base pair DNA and the arginine-rich histone tetramer (H3/H4)2. The histones bind to DNA in a 1:1 molar ration to form a stable particle which orients in an electric field with a rotational correlation time of 6.3 mus and a limitign reduced dichroism of --0.74. The complex was modeled hydrodynamically as a cylinder of dimensions 450 X 80 X 80 A containing approximately 1.5 superhelical turns. Addition of the lysinerich histones to this complex cause a condensation of the structure and results in physical properties nearly identical with those of a native nucleosomal particle.


Synthesis of des(tetrapeptide B(1-4)) and des(pentapeptide B(1-5)) human insulins. Two biologically active analogues. Two analogues of human insulin, des(tetrapeptide B(1-4))-and des (pentapeptide B(1-5))-insulin, which differ from the parent molecule in that the N-terminal tetrapeptide and pentapeptide sequences, respectively, have been eliminated, have been synthesized. The des(tetrapeptide B(1-4))-insulin shows potencies of 13 IU/mg by the mouse convulsion assay method and 7.6 IU/mg by the radioimmunoassay method. The des(pentapeptide B(1-5))-insulin possesses a potency of 1.2 IU/mg when assayed by the glucose-oxidation method in isolated fat cells and 3.7 IU/mg by the radioimmunoassay technique. The natural hormone has a potency of 23--25 IU/mg by both assay methods.


Ethidium bromide complexes with self-complementary deoxytetranucleotides. Demonstration and discussion of sequence preferences in the intercalative binding of ethidium bromide. The binding of ethidium bromide to the self-complementary deoxytetranucleotides containing guanine and cytosine bases has been studied by circular dichroism, visible absorption, and fluorescence spectroscopies. The circular dichroism spectrum of each of the four deoxytetranucleotides was measured and compared to the spectrum calculated by a nearest-neighbor approximation method which utilized the circular dichroism spectra of the deoxydinucleotides as a basis set. Reasonable agreement was obtained between the calculated and experimental spectra for three of the four deoxytetranucleotides; however, pdC-dC-dG-dG exhibited poor agreement between the actual and nearest-neighbor calculated spectra, which suggests that pdC-dC-dG-dG may exist in an unusual conformation. A nearest-neighbor method was also used to calculate the extinction coefficients for each of the deoxytetranucleotides: these values differ substantially from values recently published by Patel and Canuel [(1977), Proc. Natl. Acad. Sci. U.S.A. 74, 2624--2628] in which the method used for the determination of the extinction coefficients was not stated. The magnitude of the extinction coefficients is important in determining the stoichiometry of complex formation as well as the relative sequence preferences for ethidium binding. The visible absorption titrations, the fluorescence titrations, and the circular dichroism titrations with ethidium bromide clearly show that two ethidiums will intercalate into a (pdC-dG-dC-dG).(pdC-dG-dC-dG) double helix presumably at the two (dC-dG).(DC-dG) sequences. Results from the pdG-dC-dG-dC titrations with ethidium bromide are not as definitive. Raising the temperature from approximately 2 to 26 degrees C diminishes the strength of complex formation for the EthBr plus pdC-dG-dC-dG system and makes it more difficult to unequivocally determine the stoichiometry of the complex formation. These data thus confirm and extend our earlier observation that ethidium bromide preferentially binds to pyrimidine-purine sequences as opposed to purine-pyrimidine sequences. Experiments monitoring the binding of ethidium bromide to pdC-dC-dG-dG and pdG-dG-dC-dC indicate that ethidium will bind strongly to the (dG-dG).(dC-dC) sequence. We conclude that the relative ordering of the sequence preferences for the binding of ethidium to the three sequences available in the tetranucleotides studied is (dC-dG).(dC-dG) congruent to (dG-dG).(dC-dC) greater than (dG-dC).(dG-dC).


Photochemistry of cytosine derivatives. 1. Photochemistry of thymidylyl-(3' leads to 5')-deoxycytidine. The photochemistry of thymidylyl-(3' leads to 5')- deoxycytidine (dTpdC) was studied as a model system of adjacent thymine and cytosine bases in DNA. Acetophenonesensitized irradiation causes the cytosine moiety in dTpdC to react with the thymine moiety intramolecularly. Three unstable photoproducts are formed initially which are converted into three isomeric dinucleoside phosphates of thymine-uracil cyclobutane photodimer in a ratio of 4.2:2.2:1. Under the same irradiation condition thymidylyl-(3' leads to 5')-thymidine (dTpdT) yields two products in a ratio of 6:1. The structures of these products are established by chemical and spectroscopic methods. The major product in these reactions has been identified as the stereoisomer which has the same anti,anti relationship between the pyrimidine rings and the deoxyribose group as in the parent dinucleoside phosphates. The efficiency of the intramolecular dimerization of dTpdC is about one-third that of dTpdT. The results suggest that the cytosine base in DNA may be converted to a uracil base via photodimerization with an adjacent pyrimidine base, hydrolysis, and photoreactivation.


Photochemistry of cytosine derivatives. 2. Photohydration of cytosine derivatives. Proton magnetic resonance study on the chemical structure and property of photohydrates. Photohydrates of cytidine and cytidylic acids have been definitively characterized to be isomeric 6-hydroxy-5,6-dihydrocytosine derivatives. It has also been demonstrated by nuclear magnetic resonance spectroscopy that (1) the stereochemistry of photohydration is random, (2) the C5-H trans to the C6-OH undergoes a rapid selective exchange in the presence of proton acids, and (3) the dehydration of photohydrates is a trans-elimination. The mechanism of these processes is discussed.


Effect of inhibition of DNA synthesis on histone synthesis and deposition. We have reinvestigated the degree of coupling between DNA and histone synthesis in mammalian cells. In at least one cell line (HTC cells), the coupling is not nearly as tight as had previously been inferred from experiments with HeLa cells. The site of deposition of such histones which continue to be made in the presence of sufficient hydroxyurea to depress DNA synthesis almost totally has been studied. Deposition seems to be on material which absorbs at 260 nm. This material is not a part of the bulk chromatin and binds histone in a relatively tight manner. The possible role of such a material in histone synthesis and deposition is discussed.


Nucleosome structure of Xenopus oocyte amplified ribosomal genes. The chromatin subunit or nucleosome structure of the amplified, extrachromosomal, ribosomal genes of oocytes of the amphibian Xenopus laevis has been investigated during stages of growth when these genes are markedly changing their rates of transcriptional activity. Nucleic acid hybridization studies involving micrococcal nuclease derived monomer nucleosome DNA fragments and purified ribosomal RNAs indicate that the apparent degree of accessibility of the ribosomal genes to short-term nuclease hydrolysis varies as a function of the rate of ribosomal RNA (rRNA) transcription. However, at no stage during oocyte development are all of the amplified ribosomal genes completely accessible to nuclease hydrolysis, even in those stages with maximal rates of rRNA transcriptional activity. These results suggest that the transcriptionally active ribosomal genes of oocytes are partially, or perhaps transiently, associated with histones in the form of nuclease releasable nucleosomes but that the degree of this association may change with varying rates of rRNA synthesis. Additionally, the present data indicate that the average size of the double-stranded ribosomal DNA associated with monomer nucleosomes is the same (about 200 base pairs) in all of the oocyte stages examined regardless of the rates of rRNA synthesis in these stages.


Fluorescence quenching as an indicator for structural fluctuations in liver alcohol dehydrogenase. N-Acetyltryptophanamide (NATA), when illuminated anywhere within the 280-nm absorption band, has an emission lifetime of 3.1 ns. The tryptophan residues in liver alcohol dehydrogenase (LADH), however, when excited at 280 nm exhibit two lifetimes of r1 = 2.2 and of r2 = 5.7 ns. Excitation at 300 nm yields a single decay of 5.0 ns. It is shown that at the latter wavelength, only the two (equivalent) tryptophan residues buried within the LADH structure are excited. The reaction rate of the NATA fluorescence quenching by ionic and nonionic quenchers is practically independent of the temperature (between 5 and 41 degrees C). The same substances were used to quench the tryptophan fluorescence in LADH. Here (in the same temperature range), the quenching rate decreases drastically with a decrease in temperature. These findings are discussed in terms of conformational fluctuations in LADH, whereby the temporal movement of the polypeptide chains opens channels through which the above quencher molecules can diffuse and reach the tryptophan residues located within the enzyme structure.


Binding of bovine coagulation factor Xa to platelets. The binding of highly purified bovine coagulation factor Xa to washed bovine platelets was studied. 125I-labeled factor Xa underwent binding to a platelet receptor that became accessible only after induction of the platelet release reaction by thrombin or by the calcium ionophore A 23187. The zymogen factor X did not bind to platelets. The factor Xa binding was saturable, reversible, and correlated with the rate of thrombin formation. The number of factor Xa binding sites per platelet was 290--420 and the apparent association constant was estimated to be 2.8 x 109 to 1.0 x 1010 M-1. Diisoprophyl fluorophosphate-factor Xa bound to the same platelet receptor as factor Xa indicating that limited proteolysis of a receptor protein was not required for binding. The rate of factor Xa binding was rapid (2.1 x 10(6) to 2.9 x 10(6) M-1 s-1) and similar to that preveiously found for the rate of binding of polypeptide hormones to their receptors. Displacement of factor Xa from the platelet receptor by diisopropyl fluorophosphate-factor Xa effectively blocked thrombin formation. Low concentrations of factor Xa catalyze prothrombin activation more effectively in the presence of platelets than in the presence of phospholipid and factor V.


The histone core complex: an octamer assembled by two sets of protein-protein interactions. A protein complex, extracted from calf thymus chromatin with 2 M NaCl, pH 7.5, containing approximately equal molar ratios of histones H2A, H2B, H3, and H4, has been characterized in this study. Gel filtration, sedimentation velocity, and sedimentation equilibrium experiments demonstrate that this complex, known as the core complex, has a molecular weight near that expected for a histone octamer (108 000 for a unit containing two each of the four inner histones) and far exceeding that of a histone tetramer (54 400). This finding suggests that the histone octamer, postulated to be the fundamental histone unit in chromatin, is stable in 2 M NaCl, pH 7.5, in the absence of DNA or chemical cross-linking reagents. In the second part of this study, we demonstrate that the bonds maintaining the octameric complex in 2 M NaCl are weak and distinctly different from the forces stabilizing the H2A-H2B dimer or H3-H4 tetramer. The octamer is dissociated into two H2A-H2B dimers and one H3-H4 tetramer by (i) increasing temperature; (ii) decreasing NaCl concentration; (iii) adding low concentrations of urea or guanidine hydrochloride; and (iv) lowering the pH below 7 or raising it above 10. These findings indicate that the octamer is assembled by two sets-of protein-protein interactions. The first set involves mostly hydrophobic interactions and yields the H2A-H2B dimer and the H3-H4 tetramer subunits. The second set involves the weak association of one H3-H4 tetramer with two H2A-H2B dimers to form an octamer. We suggest that these weak interactions might be derived predominantly from histidine-lysine or histidine-tyrosine hydrogen bonds between the dimer and tetramer subunits.


Equilibrium and kinetic studies of the binding of Lens culinaris lectin to rabbit erythrocytes by a quantitative fluorometric method. The binding of Lens culinaris lectin to the receptors of rabbit erythrocytes was studied by a quantitative fluorometric method using the fluoresceinated conjugate. Equilibrium data showed the presence of 1.2 x 10(6) receptors/cell with an association constant varying from 8 to 3 x 10(6) M-1 over a temperature range of 5 to 37 degrees C. The binding reaction is exothermic with a deltaH = -4.6 kcal/mol and a deltaS = + 15 eu. It appears that most of the bound lectin molecules had only a single site occupied, the agglutination being due to a very low fraction (0.0003--0.0056) of lectin molecules with two sites occupied. The specificity of the reaction was demonstrated through its inhibition by nonfluoresceinated LL and by the specific sugars methyl alpha-D-glucoside and methyl alpha-D-mannoside. From the inhibition curves an association constant of 4 x 10(2) M-1 was calculated for methyl alpha-d-glucoside and 9 x 10(2) for methyl alpha-D-mannoside. The association and dissociation rate constants of the binding reaction are, respectively, in the range of 3--10 x 103 and 3--33 x 10(-4) M-1 s-1 in the temperature range of 5 to 37 degrees C. The activation energies of the forward and reverse reactions are 7 and 13 kcal/mol, respectively. The association constants and the binding enthalpy calculated from the activation energies were fully consistent with those obtained by equilibrium measurements.


Effect of cholesterol on the molecular motion in the hydrocarbon region of lecithin bilayers studied by nanosecond fluorescence techniques. Effects of cholesterol on the dynamic structure of the hydrocarbon region of dipalmitoyllecithin vesicles were examined. Decays of the emission anisotropy and the fluorescence intensity of 1,6-diphenyl-1,3,5-hexatriene embedded in lecithin-cholesterol vesicles were measured over a temperature range of 10--60 degrees C. The emission anisotropy decreased rapidly with time and then leveled off. The rotational motion of the probe was analyzed by a model of wobbling diffusion confined in a cone. Cholesterol (10--50 mol%) decreased the cone angle in the liquid-crystalline phase and increased it in the gel phase. In the presence of 33 mol% cholesterol, the wobbling diffusion constant increased in the gel phase and changed little in the liquid-crystalline phase. The viscosity in the cone decreased in the gel phase and remained almost unchanged in the liquid-crystalline phase in the presence of 33 mol% cholesterol. The total fluorescence intensity followed a singel exponential decay independently of the cholesterol content 0--50 mol%.


Structural requirements of alkyl acyldithiocarbazates for the uncoupling of oxidative phosphorylation in mitochondria. A structure-activity relationship study on the uncoupling of alkyl acyldithiocarbazates was carried out. Greater activity was observed with increasing alkyl chain length, the optimum being C9. A further increase in alkyl chain length caused a decrease in the activity. Thione-thiol tautomeric forms with a dissociable proton were dound to be of primary importance for the uncoupling and the role of the acyl group was auxiliary. The reactivity of the SH group of alkyl acyldithiocarbazates with an SH-reagent was very low. These compounds facilitated the valinomycin-induced swelling of non-respiring mitochondria and non-sonicated lecithin liposomes in isotonic potassium acetate solution.


The accumulation of neutral red in illuminated thylakoids. Thylakoids isolated from spinach (Spinacia oleracea L.) bind only a small fraction of neutral red in the dark whereas they accumulate large amounts of the protonated dye in their inner space under light. Light-induced neutral red uptake depends on the size of the proton gradient across the thylakoid membrane but does not follow the mechanism established for amines. Instead, the correlation between pH gradient and neutral red uptake can be predicted quantitatively assuming that protonated neutral red is accumulated mainly as dimer species. Under appropriate conditions, accumulation of protonated neutral red in the inner thylakoid space is proportional to an absorbance increase at 520 nm. This 520-nm change can be used for the continuous measurement of pH changes in thylakoids during steady-state illumination.


Functional homogeneity of P-700 in cyclic and non-cyclic electron transport reactions in thylakoids. The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N',N'-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP+ as acceptor, the illuminated steady-state concentration of P-700+ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo. The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg2+ or Ca2+, had no effect on the signal amplitude during cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700+ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg+. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.


Cyclic electron transport in isolated intact chloroplasts. Further studies with antimycin. Antimycin has been used to study the role of cyclic electron transport in isolated intact chloroplasts maintained under aerobic conditions. At all light intensities, antimycin inhibits CO2 fixation when assay conditions are optimal. When turnover of the Calvin cycle is inhibited, antimycin stimulates bicarbonate-dependent O2 evolution. Energy-dependent processes such as chlorophyll a and 9-aminoacridine fluorescence quenching, and light-scattering (apparent absorption) changes are inhibited by antimycin. The results suggest that cyclic electron transport contributes to photophosphorylation under aerobic conditions and is obligatory as a source of ATP during the most active periods of CO2 fixation in vivo. Cyclic electron transport can be stimulated either by inhibiting Photosystem II activity or increasing the turnover of Photosystem I relative to Photosystem II. These effects are interpreted in terms of the need for correct redox poising of carriers in the pathway in order to sustain maximum rates of cyclic electron flow. Binding studies indicate the presence of a high affinity antimycin binding site on chloroplast membranes. The stoichiometry and dissociation constant of the high affinity site are consistent with the idea that antimycin inhibits cyclic electron transport by binding to a b-type cytochrome in the thylakoid membrane.


Chloroplast culture: the chlorophyll repair potential of mature chloroplasts incubated in a simple medium. The chlorophyll repair potential of mature Cucumis chloroplasts incubated in a simple Tris-HCl/sucrose medium is described. The chloroplasts were isolated from green, fully expanded Cucumis cotyledons which were capable of chlorophyll repair. This was evidenced by a functional chlorophyll biosynthetic pathway in the mature tissue. The biosynthesis of protocholorphyllide from exogenous delta-aminolevulinic acid was used as a marker for the operation of the chlorophyll biosynthetic chain between delta-aminolevulinic acid and protochlorophyllide. The conversion of exogenous protochlorophyllide into chlorophyll a was used as a marker for the operation of the chlorophyll pathway beyond protochlorophyllide. It appeared from these studies that contrary to published reports, unfortified fully developed Cucumis chloroplasts incubated in Tris-HCl/sucrose without the addition of cofactors exhibited a partial and limited chlorophyll repair capability. Their net tetrapyrrole biosynthetic competence from delta-aminolevulinic acid was confined to the accumulation of coproporphyrin. No net tetrapyrrole biosynthesis beyond coproporphyrin was observed. However, the plastids were capable of incorporating small amounts of delta-amino[4-(14)C]levulinic acid into [14C]protochlorophyllide but were incapable of converting exogenous protochlorophyllide into chlorophyll. After prolonged incubation of the unfortified chloroplasts in the dark, a fluorescent protochlorophyllide-like compound accumulated. This compound [Cp (E430-F631) was shown to be neither protochlorophyllide nor zinc-prototochlorophyllide but and a fluorescence emission maximum at 631 nm (F631) in methanol/acetone (4 : 1, v/v). Cp(E430-F631) WAS SHOWN TO BE NEITHER PROTOCHLOROPHYLLIDE NOR ZINC-PROTOTOCHLOROPHYLLIDE BUT an enzymatic degradation product of chlorophyll. The exact chemical identity of this compound has not yet been determined.


Membrane surface potential and the reactivity of the system II primary electron acceptor to charged electron carriers in the medium. A hypothesis is proposed to explain the changes in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase. Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149--166). Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.


Circular dichroism of squid rhodopsin and its intermediates. Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates was measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the alpha- and beta-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at alpha-band and 348 nm at beta-band) as well as at room temperature (CD maxima: 474 nm at alpha-band and 347 nm at beta-band). The rotational strength of the alpha-band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at alpha-band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may de derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, The CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.


Redox state of the partially reduced cytochrome aa3- cyanide complex. The low-spin ferric cyanide complex of beef heart cytochrome aa3 can be partially reduced by stoichiometric additions of ferrous cytochrome c or by similar additions of N,N,N',N'-tetramethyl-p-phenylene diamine. In both cases the initial ratio of cytochrome c oxidized: cytochrome a reduced or Wurster's Blue: cytochrome a reduced approximates the value 2. It is concluded that the binding of a single HCN prevents the reduction of both cytochrome a3 and its associated EPR-invisible Cu atom.


The influence of the lipid on the water permeability of artificial membranes. The water permeabilities of artificial membranes formed formed from various monoglycerides or phospholipids in alkane solvents have been measured using an osmotic method, and it has been shown that the permeability depends upon the type of lipid used. For monoglycerides, the permeability was found to increase with the unsaturation and decrease with the length of the acyl chain. Membranes formed from either egg phosphatidylcholine or dioleyl phosphatidylcholine had an osmotic permeability coefficient of approx. 35--40 micrometer/s at 25 degrees C; with sphingomyelin as the membrane lipid, the permeability was an order of magnitude lower than that for phosphatidylcholine. It is suggested that the water permeabilities of biological membranes might be partly controlled by the types of lipid present.


Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization. The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive 125-I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-aminopyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, aparticular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.


Testing transport models with substrates and reversible inhibitors. The kinetic behavior of five models for biological transport, only one of which is based on the classical carrier mechanism, is investigated. All give hyperbolic substrate saturation curves in accord with experimental observations on many systems. Several simple kinetic tests with substrates and competitive inhibitors serve to exclude or confirm proposed models. The tests involve measuring rates of efflux of radioactive substrate in the presence of (i) a competitive inhibitor outside the cell; (ii) inhibitor inside and outside; and (iii) unlabeled substrate outside. Rules for testing hypothetical mechanisms are presented in tables which may be consulted directly, disregarding the mathematical derivation.


Localization of protein-bound carbohydrate residues on the cytoplasmic surface of rough and smooth microsomes and Golgi vesicles from rat liver. Rough and smooth microsomes and Golgi membranes isolated from rat liver were treated with proteolytic enzymes under conditions which removed 30--40% of the surface proteins without seriously disrupting the membrane structure. This treatment also removed 40--60% of protein-bound mannose, galactose and glucosamine. When protease treatment was combined with neuraminidase treatment, 80% of the sialic acid was removed from intact rough microsomal and Golgi vesicles and about half of the sialic acid of smooth microsomes was solubilized. It appears that half, or probably more, of the membrane glycoproteins are associated with the cytoplasmic surface of these membranes.


Distribution of osmotic flow in stomach and gallbladder. Stomach and gallbladder actively transport fluids which are nearly isotonic to plasma. Consideration of the measured areas of the appropriate transporting surfaces gives a more realistic view of the osmotic gradient required to account for the observed net flow of water. Simple osmosis may be adequate if the transporting membrane has an osmotic permeability in the range observed for synthetic lecithin-cholesterol bilayer membranes.


The effect of cholesterol on the structure of phosphatidylcholine bilayers. The effect of cholesterol on the structure of phosphatidylcholine bilayer was investigated by X-ray diffraction methods. Electron density profiles at 5 A resolution along with chain tilt and chain packing parameters were obtained and compared for phosphatidylcholine/cholesterol bilayers and for pure phosphatidylcholine bilayers in both the gel and liquid crystalline states. The cholesterol in the bilayers was localized by noting the position of discrete elevations in the electron density profiles. Cholesterol can either increase or decrease the width of the bilayer depending on the physical state and chain length of the lipid before the introduction of cholesterol. For saturated phosphatidylcholines containing 12--16 carbons per chain, cholesterol increases the width of the bilayer as it removes the chain tilt from gel state lipids or increases the trans conformations of the chains for liquid crystalline lipids. However, cholesterol reduces the width of 18 carbon chain bilayers below the phase transition temperature as the long phospholipid chains must deform or kink to accomodate the significantly shorter cholesterol molecule. Although cholesterol has a marked effect on hydrocarbon chain organization, it was found that, within the resolution limits of the data, the phosphatidylcholine head group conformation is unchanged by the addition of cholesterol to the bilayer. The head group is oriented parallel to the plane of the bilayer for phosphatidylcholine in the gel and liquid crystalline states and this orientation is not changed by the addition of cholesterol.


Effects in adipocytes of diamide on GSH levels, glucose uptake and cell integrity. Concentrations of insulin and chemical agents (H2O2, vitamine K-5) which stimulate hexose transport in fat cells do not alter the cellular levels of glutathione (reduced form; GSH). Diamide, another agent used in studies of insulin action, markedly reduces GSH levels and increases the movement of sugar into the cell. However, unlike insulin, H2O2 or vitamin K-5, diamide causes a change in the permeability of fat cells that allows entry of compounds (insulin, sucrose, L-glucose) which are normally excluded by the plasma membrane. Moreover, the accelerated rate of methylglucose uptake produced by diamide treatment is not inhibited by cytochalasin B, an agent that blocks basal and insulin-stimulated methylglucose transport. These results indicate that diamide does not cause a stimulation of the glucose transport system and should not be used (or used with caution) in transport studies. Furthermore, oxidation of GSH does not appear to be necessary for the stimulation of hexose transport in adipocytes by insulin, H2O2 or vitamin K-5.


Affinity chromatography of the phosphatidylcholine exchange protein from bovine liver. Affinity chromatography has been used to purify the phosphatidylcholine exchange protein from bovine liver. The affinity resin consisted of 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine linked to AH-Sepharose 4 B via the carboxyl group. Application of a crude exchange protein fraction to the affinity column resulted in a complete adsorption of the phosphatidylcholine exchange protein. The exchange protein eluted with a buffer containing 0.15% sodium deoxycholate. The most active fraction was 130-fold purified and accounted for 62% of the activity.


Lateral compressibility of lipid mono- and bilayers. Theory of membrane permeability. The passive sodium permeability of pure lipid vesicles and dispersions has a large peak at the bilayer phase transition temperature. We discuss this anomaly in terms of density fluctuations, which can open up cavities in the headgroup region into which small ions can enter, and which may be large if bilayer conditions at the melting point are similar to those near the critical point which seems to exist in monolayers. We present two arguments, one thermodynamic and one microscopic, which suggest that the permeability is proportional to the lateral compressibility. We then calculate the lateral compressibility for two previously published theoretical models and compare the results with experiment.


Particle segregation in chromaffin granule membranes by forced physical contact. Bovine chromaffin granules were exposed to different isotonic non-ionic and ionic solutions (sucrose; Ca2+- and Mg2+-free phosphate-buffered saline; Tris-HCl + NaCl; Ca2+- and Mg2+-free phosphate-buffered saline + sucrose; Tris-HCl + sucrose) at pH 7 and then frozen either in suspension or as firm pellets. Freezing was performed without prefixation or antifreeze treatments either by 'standard' techniques (approx. 1 mm3 suspended or pelleted material on gold specimen supports dipped into liquid Freon) or with increased cooling rates by spraying suspensions into liquid propane ('spray-freezing'). Regardless of the freezing method, membrane-intercalated particles were always randomly distributed when chromaffin granules were frozen in suspension. In contrast, forced physical contact between granules produced by centrifugation (12000 X g, 25 min) provoked dispersal of membrane-intercalated particles, but only in the presence of ions. Sucrose or EDTA in an ionic environment had no inhibitory effect. The following conclusions are derived: (1) Even below the reported phase transition region particle clustering is possible. (2) Chromaffin granule membranes are not liable to thermotropic segregation of membrane-intercalated particles. (3) Although the low freezing rates of 'standard' freezing techniques produce large-scale segregation artefacts (by which suspended chromaffin granules are pushed together within the segregated solute) this does not result in intramembraneous particle segregation. (4) Forced physical contact produces a Ca2+-independent particle segregation, but only when repulsive electrostatic forces of membrane components are partially screened in an ionic environment. (5) This does not invalidate results obtained by others, showing Ca2+-mediated chromaffin granules agglomeration and segregation of membrane-intercalated particles, but it might indicate the occurrence of another, not directly Ca2+-dependent particle segregation mechanism in a prefusional stage of close membrane-to-membrane contact during exocytosis.


The orientation of D-beta-hydroxybutyrate dehydrogenase in the mitochondrial inner membrane. D-beta-Hydroxybutyrate dehydrogenase of beef heart mitochondria is a lipid-requiring enzyme, bound to the inner membrane. The orientation of this enzyme in the membrane has been studied by comparing the characteristics of the enzyme in mitochondria and 'inside-out' submitochondrial vesicles. We observe that the enzymic activity is (1) latent in intact mitochondria; (2) relatively stable to trypsin digestion in mitochondria but rapidly inactivated in submitochondrial vesicles by this treatment; and (3) released more rapidly from submitochondrial vesicles by phospholipase A2 digestion than from mitochondria. Conclusive evidence that D-beta-hydroxybutyrate dehydrogenase is localized on the matrix face of the mitochondrial inner membrane is provided by the correlation that the enzyme is released from submitochondrial vesicles before the membrane becomes leaky to cytochrome c. The arrangement of D-beta-hydroxybutyrate dehydrogenase in the membrane is discussed within a generalized classification of the orientation of proteins in membranes. The evidence indicates that D-beta-hydroxybutyrate dehydrogenase is an amphipathic molecule and as such is inlaid in the membrane, i.e. the enzyme is partially inserted into the hydrophobic milieu of the membrane, with the polar, functional end extending into the aqueous milieu.


Effect of anions of potassium self-exchange in ascites tumor cells. Potassium self-exchange in ascites cells was partially suppressed when chloride in the incubation medium was replaced by bromide, and completely abolished in iodide-, nitrate- or sulfate-containing saline. Thus, it appears that potassium self-exchange activity is anion-dependent.


Fluorescent probes in model membranes. II. Monolayer studies of 2,2'-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and N-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid. Film studies at the air-water interface have been carried out for pure films of 2,2'-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxyequilenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as "non-perturbing" and should be retained in condensed lipid phases. ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.


The adsorption of divalent cations to phosphatidylcholine bilayer membranes. Electrophoretic mobility and 31P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations of phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the 31P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the 31P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Sr2+, Ba2+. If we assume that a lipid molecule occupies an area of 60 A2 and that there is a 1 : 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M.


Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice. Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanol-treated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.


The composition and biosynthesis of the glycoproteins and glycolipids of the rabbit small-intestinal brush border. 1. The glycoprotein and glycolipid composition of isolated rabbit small-intestinal brush borders has been studied. 2. The total glycoprotein fraction contains an average 95 microgram carbohydrate per mg protein, composed of mannose, galactose, fucose, N-acetylglucosamine and N-acetylgalactosamine. Glucose is also present but sialic acid is absent. 3. The isolated glycolipids include ceramide lactoside, ceramide trihexoside and two N-acetylglucosamine-containing glycolipids. Sialic acid containing glycolipid (gangliosides) is present only in trace quantities. 4. The biosynthesis of the brush border-bound glycoproteins and glycolipids has been studied following intraperitoneal injection with D-[1-14C]glucosamine and isolation of the brush borders at intervals between 3 and 24 h. 5. The total glycoprotein fraction labels maximally 7.5 h after injection and subsequently exhibits an exponential loss of radioactivity with a half-life of 11.2 h. The labelling kinetics of one of the glucosamine-containing glycolipids is similar to that of the glycoproteins in that it labels maximally between 7.5 and 12 h, but the second glucosamine-containing glycolipid labels later at approximately 18 h. These results indicate that the glycoproteins and glycolipids are actively synthesized and degraded within the mature small intestinal enterocyte and that individual glycolipids turn over independently.


Synaptic membranes from Torpedo marmorata electric organ. 1. Separation and analysis of nicotinic acetylcholine receptor- and acetylcholinesterase-containing membrane vesicles using aqueous two-phase systems. Two-phase systems consisting of water, dextran and poly(ethylene glycol) have been used for partition of membranes obtained from Torpedo marmorata electric organ. The partition behaviour of the membranes could be adjusted by using a polymer with covalently-bound charged groups. By using this method, the membranes were divided into several fractions which were analyzed for nicotinic acetylcholine receptor and acetylcholinesterase content. It was found that nicotinic receptor-enriched membranes were separated from those containing esterase in a single partition step. Receptor-enriched membranes obtained by gradient centrifugation could be further separated into two receptor fractions by the two-phase technique. The results also reveal at least two types of acetylcholinesterase-rich membranes.


Cholesterol uptake is dependent on membrane fluidity in mycoplasmas. The transfer of elaidate-enriched Acholeplasma laidlawii cells in culture from 37 degrees C to 4 degrees C virtually arrested exogenous cholesterol incorporation into the cell membrane. Cholesterol uptake continued, though at a slower rate, in oleate-enriched A. laidlawii cells undergoing similar temperature shift-down. It is concluded that the incorporation of exogenous cholesterol into the cell membrane of living mycoplasmas is rapid when the membrane lipid bilayer is in the liquid-crystalline state and very slow when the lipid bilayer is in the gel state.


Evidence for isotropic motion of phospholipids in liver microsomal membranes. A 31P NMR study. 1. The motional properties of phospholipids in bovine and rat liver microsomes and aqueous dispersions of the extracted lipids have been investigated employing 31 P NMR techniques. 2. The 31P NMR spectra obtained from the microsomes indicate that a considerable portion of the constituent phospholipids experience isotropic motion on the NMR timescale (10(-5) s). This is in strong contrast to the spectra obtained from aqueous dispersions of the extracted lipids, which display the characteristic lineshape associated with liquid crystalline phospholipids in (large) bilayer structures, which experience restricted anisotropic motion. 3. Evidence is presented which strongly suggests that the isotropic motion of microsomal phospholipids does not arise from tumbling of the microsomal vesicles or from lateral diffusion of phospholipids around these vesicles.  4. These results are discussed in terms of possible transitory formation of intramembrane non-bilayer lipid configurations, with which the bulk (bilayer) phospholipids are in rapid exchange.


Fusion of isolated myoblast plasma membranes. An approach to the mechanism. Fusion of plasma membranes isolated from myoblasts grown in culture has been investigated. 1. Membrane fusion was specifically dependent of Ca2+ at physiological concentrations. However, at higher concentrations of cations, fusion could be triggered not only by Ca2+, but by Mg2+ and Sr2+ as well. 2. The amount of fusion was directly proportional to temperature. 3. Fusion was found to depend on the state of maturation of the myoblast membranes. 4. Experiments with chemically and enzymatically modified membranes and with membranes derived from myoblasts grown in the presence of inhibitors of protein biosynthesis suggest the participation of proteinaceous membrane components in the fusion mechanism.


The effect of a disulfonic acid stilbene on proximal cell membrane potential in Necturus kidney. The effects of 0.5 mM 4-acetamido-4'-isothiocyano-stilbene-2,2' disulfonic acid on the electrical properties of the peritubular membrane were studied in the proximal tubule of the perfused Necturus kidney. The addition of stilbene isothiocyanate disulfonic acid in peritubular perfusate resulted in an average 4.5 mV hyperpolarization with no detectable changes of peritubular membrane input conductance. The depolarization elicited by high-K media was enhanced by 18% in the presence of stilbene isothiocyanate disulfonic acid, an observation indicating that the inhibitor increased the contribution of potassium to membrane potential, presumably by decreasing anionic permeabilities. The hyperpolarizing effect of stilbene isothiocyanate disulfonic acid was abolished when peritubular bicarbonate was removed from the medium and isoosmotically replaced by chloride. These data suggest that (a) intracellular bicarbonate activity is higher than that predicted from passive distribution, (b) stilbene isothiocyanate disulfonic acid decreases P HCO3, thus hyperpolarizing the membrane, (c) chloride distribution appears to be passive when bicarbonate is removed from the peritubular perfusate. The state of Cl distribution when extracellular bicarbonate is at physiologic concentration cannot be assessed from the present data.


Separation of the proteins of bovine milk-fat globule membrane by electrofocusing. The proteins of milk-fat globule membrane have been separated by electrofocusing on both the analytical and preparative scale. Over forty separated proteins of the membrane can be identified after electrofocusing in the presence of urea, Triton X-100 and mercaptoethanol with apparent isoelectric points between pH 5.0 and 9.0. At least eight of these proteins appear to contain carbohydrate. After separation by electrofocusing the samples have been further analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Some of the proteins previously identified as single bands by electrophoresis in SDS are resolved into several components by electrofocusing. The major components of milk-fat globule membrane are a glycoprotein of 67 000 daltons, with an apparent isoelectric point of 5.55, and a protein of 155 000 daltons with an isoelectric point of 7.6. Partially purified fractions of the major proteins and glycoproteins can be obtained after preparative electrofocusing in flat-beds of Sephadex G-75.


Fluorescence of 1,8-anilinonaphthalene sulfonic acid bound to proteins and to lipids of sarcolemma. Relative contributions to fluorescnece of 1,8-anilinonaphthalene sulfonic acid-sarcolemma by 1,8-anilinonaphthalene sulfonic acid bound to proteins and to lipid were assessed on the basis of fluorescence lifetime and steady-state emission spectra. The lifetime spectrum of lipid-poor proteins prepared from sarcolemma had a major time constant of 16 ns; that of lipids extracted from sarcolemma had a major time constant of 7 ns and a minor one of 4 ns. In sarcolemma, components having all three time constants were observed with weighting factors 0.33, 0.6 and 0.07, respectively, indicating the fraction of 1,8-anilinonaphthalene sulfonic acid bound to protein and to lipid. Steady-state emission spectra of 1,8-anilinonaphthalene sulfonic acid-sarcolemma were resolved into and resynthesized from contributions by 1,8-anilinonaphthalene sulfonic acid bound to proteins and to lipids. The latter was at least two thirds of total 1,8-anilinonaphthalene sulfonic acid bound. Results of analyses of spectra obtained with various concentrations of sarcolemma and 1,8-anilinonaphthalene sulfonic acid suggest that the proteins have a higher affinity, but only about half the binding capacity for 1,8-anilinonaphthalene sulfonic acid as do lipids.


Effect of glycophorin incorporation on the physico-chemical properties of phospholipid bilayers. 1. The thermotropic behaviour of phospholipid molecules in reconstituted glycophorin-containing vesicles has been investigated by means of differential scanning calorimetry. Each glycophorin molecule is able to perturb the properties of 80--100 phospholipid molecules in such a way that these lipid molecules no longer participate in the cooperative gel to liquid-crystalline phase transition. This number of perturbed phospholipid molecules was discovered to be independent of the lipid charge. 2. By means of freeze-facture electron microscopy it could be demonstrated that glycophorin is not excluded from the solid lipid phase upon cooling the lipids below their gel to liquid-crystalline phase transition temperature. In mixtures of phosphatidylcholines which show solid-solid immiscibility, glycophorin is preferentially associated with the lower-melting lipid component upon phase separation, as could be demonstrated by both differential scanning calorimetry and freeze-fracture electron microscopy. 3. The effect of glycophorin on the mobility of phospholipids has been investigated by means of 31 P NMR. Glycophorin, incorporated into sonicated vesicles of dioleoylphosphatidic acid, is able to immobilize nine lipid molecules very strongly in their phosphate region. Evidence for an electrostatic inter-action between the protein and this negatively charged phospholipid has been presented. 4. The presence of glycophorin causes discontinuities in the lipid bilayer. This results in higher susceptibility of the bilayer towards attack by lipolytic enzymes and in enhanced membrane permeability.


Enhanced release of nascent single strands from DNA synthesized in the presence of arabinosylcytosine. Arabinosylcytosine at a 1 . 10(-4) molar concentration inhibited thymidine incorporation into DNA by more than 95%. In sucrose gradients the labelled dThd was predominantly found in short DNA chains. Labelled arabinosylcytosince (aC) was incorporated into DNA, as was labelled dThd, indicating that it causes a preferential inhibition of the chain polymerization rather than termination of nascent chains. This was confirmed by the observation of the conversion of short chains to normal size DNA molecules in chase experiments. In the absence of NaCl and at neutral pH a release of more than 50% of the nascent label as single strands was repeatedly observed upon sucrose gradient centrifugation. This release could be significantly reduced by 1 M NaCl, indicating that in the presence of aC the in vivo structure was preserved. This was further confirmed by the fact that aC did not cause detachment of DNA from a rapidly sedimenting nuclear structure. Based on these results, a replication model slightly different from the one suggested by Okazaki, is proposed, in which initiations of new nascent chains can occur at certain distances ahead of the replication fork.


Stability of DNA replicating activity in permeabilized mouse cells during preincubation. When permeabilized cells, treated with detergent and made permeable to the nucleoside triphosphates, were preincubated briefly without nucleoside triphosphates, the activity of DNA replication was lost rapidly. This loss of DNA replicating activity was prevented when the mixture of nucleoside triphosphates (5 mM ATP and 0.1 mM each of dATP, dGTP, dCTP and TTP, the same concentrations contained in reaction mixture) was added to the permeabilized cells during the incubation. Each of deoxyribonuclesode triphosphates or ribonucleoside triphosphates, when added at 5 mM, was effective to varying degrees, but ATP was the most effective. These results suggests that there exists a process or factor(s) that requires ATP for DNA replication in mammalian cells, and that its decay during the preincubation could be prevented by ATP.


An easy and efficient procedure for the isolation of pure DNA restriction fragments from agarose gels. A new procedure is developed to isolate DNA from agarose gels. Using a kind of blotting technique, DNA is isolated from the gel. It is shown that the isolated DNA can be used for fragmentation by restriction endonucleases, synthesis of complementary RNA by DNA-dependent RNA polymerase from Escherichia coli and nick translation. The procedure gives a high recovery and is easy to perform.


Mapping of restriction sites in the transforming HpaI-E fragment of adenovirus type 5 DNA. Adenovirus type 5 (Ad5) DNA was degraded with endo R . HpaI; the left-terminal fragment, HpaI-E has recently been shown to be the smallest segment of Ad5 DNA, that can transform non-permissive cells. This fragment was labelled at its termini by limited exonuclease III digestion followed by repair synthesis with DNA polymerase and alpha-32P-labelled deoxynucleoside triphosphates. It was then further digested with each of the restriction endonucleases HpaII, HaeIII, AluI, HinfI and TaqI; the cleavage products thus obtained were ordered into a physical map.


Sequence studies on mouse L-cell satellite DNA by base-specific degradation with T4 endonuclease IV. The base sequence of mouse L-cell satellite DNA was investigated by degradation of the two separated complementary strands with the base specific enzyme, T4 endonuclease IV. Digestion of the heavy strand DNA released a limited number of oligonucleotides which were separated by ionophoresis/homochromatography, isolated, and sequenced by the 'wandering spot' method. The light strand DNA was resistant to digestion with T4 endonuclease IV and no detectable amounts of oligonucleotides were released. The oligonucleotides obtained from the heavy strand were related in sequence, indicating that mouse satellite DNA derived from a short tandemly repeated sequence. The sequence of part of the original repeat unit is proposed to be C-A-T-T-T-T-T-C. Five major oligonucleotides were identified, all of which differ from the proposed original sequence by single base changes. The five major oligonucleotides occur with about equal frequency and together comprise approximately 50% of the oligonucleotides released by T4 endonuclease IV from the heavy strand DNA. In addition to the five major oligonucleotides, several oligonucleotides were found to occur in lesser amounts. Since these oligonucleotides are related to the major oligonucleotides, it is likely that they have arisen from them by mutation.


ATP-independent DNA synthesis in vaccinia-infected L cells. Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl2. DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes.


Inhibition of translation of lens mRNAs in a messenger dependent reticulocyte lysate by cap analogues. The nuclease treated reticulocyte lysate forms a highly efficient and completely mRNA-dependent cell-free system. In this system the functioning of the cap on eukaryotic mRNAs was explored by blocking cap recognition with cap analogues. Translation of capped mRNAs was severely inhibited, while translation of uncapped mRNAs was unaffected. It is concluded that this cell-free system can be used for screening cap dependence in the translation of specific mRNAs, like calf lens mRNAs. At 1.2 mM m7G5'p, 0.16 mM m7G5'pp or 0.16 m7G5'ppp5'G, translation of all lens mRNAs was totally inhibited. At lower concentrations the sensitivity to cap analogues was different for the various species of lens crystallin messenger. gamma-Crystallin mRNA showed relatively the lowest response. The translation of added polyribosomes was also inhibited by the cap analogue. It is concluded that translation of all crystallin messengers is cap-dependent.


Purification of class A, B, and C DNA-dependent RNA polymerases from chicken embryos. Crude nuclei were isolated from trunks of 13-day-old chicken embryos under conditions which prevent leakage of RNA polymerases from nuclei. RNA polymerases were solubilized by subsequent incubation in alkaline buffer and sonication at high salt concentration. Purification of RNA polymerases A, B, and C was achieved by conventional column chromatographic procedures. RNA polymerase B was freed from an UTP:polynucleotidyl exotransferase by chromatography on a tRNA-Sepharose column. Purified RNA polymerase A contained six putative subunits with molecular weights 190 000 (A1), 117 000 (A2), 57 000 (A3), 50 000 (A4), 25 000 (A5), 19 000 (A6); RNA polymerase B contained eight putative subunits with molecular weights 98 000 (B2'), 86 000 (B2''), 155 000 (B3), 44 000 (B4), 31 000 (B5), 28 000 (B6), 26 000 (B7), 19 000 (B8); RNA polymerase C contained nine putative subunits with molecular weights 170 000 (C1), 117 000 (C2), 84 000 (C3), 60 000 (C4), 49 000 (C5), 36 000 (C6), 33 000 (C7), 22 000 (C8), 19 000 (C9).


Effects of aminooxyacetate, alanine and other amino acids on protein synthesis in isolated rat hepatocytes. Protein synthesis in isolated rat hepatocytes has been measured by the incorporation of [14C]valine at high concentration and constant specific activity (5 mmol/l and 315 muCi/l). Protein synthesis was stimulated by the addition of an amino acid mixture, and by a number of individual amino acids alone, most notably alanine. Energy substrates (lactate, pyruvate) stimulated protein synthesis to the same extent as alanine, suggesting that a major part of the amino acid effect could be due to the provision of energy. Aminooxyacetate, an inhibitor of glutamate transaminases, inhibited protein synthesis strongly (70%), and abolished the stimulatory effects of alanine and energy substrates. This could indicate that hepatocytic protein synthesis is subject to positive control by a transamination-dependent agent.


Regulation of protein synthesis by hemin: effect of dithiothreitol on the formation and activity of the hemin-controlled translational repressor. Previous studies have demonstrated that the hemin-controlled translational repressor (HCR), a high molecular weight protein inhibitor of polypeptide chain initiation in rabbit reticulocyte lysate, is formed from a presynthesized prorepressor over a period of 12--18 h in three stages denoted reversible, intermediate, and irreversible. The prorepressor can, however, be completely converted to irreversible HCR within 2 min by incubation with such sulfhydryl reagents as N-ethylmaleimide. The results in this report demonstrate that dithiothreitol, which stabilizes thiol groups, will, like hemin, prevent the conversion of the prorepressor to HCR and will inactivate reversible HCR. Unlike hemin, dithiothreitol also inactives the intermediate form of HCR. Neither dithiothreitol nor hemin has any effect on the activity of irreversible HCR. Since the prorepressor used in these experiments had been separated from the supernatant factor (a soluble protein that reverses the inhibition of protein synthesis due to HCR), the effect of dithiothreitol and of hemin is independent of this factor and may be mediated by direct interaction with the prorepressor and HCR. Dithioerythritol, the erythro isomer of dithiothreitol, is as effective as dithiothreitol in preventing the formation of HCR, whereas glutathione and beta-mercaptoethanol have little or no effect.


Control of protein synthesis by hemin. An association between the formation of the hemin-controlled translational repressor and the phosphorylation of a 100 000 molecular weight protein. The control of protein synthesis by hemin in rabbit reticulocytes is mediated by the formation of a high molecular weight protein inhibitor of polypeptide chain initiation, termed the hemin-controlled translational repressor, from a presynthesized prorepressor. The prorepressor, purified approx. 600-fold, was used to study the mechanism of hemin-controlled translational repressor formation. When the prorepressor is converted to the hemin-controlled translational repressor, either by prolonged warming in the absence of hemin or by incubation with N-ethylmaleimide for 5 min, and then incubated briefly with [gamma-32P]-ATP and Mg2+, a protein that migrates as a 100 000 molecular weight component on sodium dodecyl sulfate-polyacrylamide gels becomes phosphorylated. The extent of phosphorylation of this component is directly proportional to the amount of prorepressor converted to the hemin-controlled translational repressor. In addition, the 100 000 molecular weight protein is not labeled when phosphorylation is attempted with the prorepressor or prorepressor warmed in the presence of hemin, indicating that the protein kinase responsible is probably the hemin-controlled translational repressor. Since the 100 000 molecular protein copurifies with the prorepressor and since the phosphorylation reaction is very rapid (50% complete within 30 s at 34 degrees C), relatively insensitive to dilution, and behaves like an intramolecular reaction, the data suggest that the hemin-controlled translational repressor, once activated, may autophosphorylate a 100 000 molecular weight subunit of itself. Approx. 5 mol phosphate are incorporated per mol of 100 000 molecular weight protein, when the prorepressor is completely converted to the hemin-controlled translational repressor by N-ethylmaleimide. Neither the rate of conversion of prorepressor to the hemin-controlled translational repressor nor the subsequent phosphorylation of the 100 000 molecular weight protein is enhanced by cyclic AMP or reduced by incubation with 3':5'-cyclic nucleotide phosphodiesterase, indicating that cyclic AMP plays no role in hemin-controlled translational repressor formation.


Effect of hypophysectomy on persistence of methylated purines in rat liver deoxyribonucleic acid after administration of dimethylnitrosamine. The formation of methylated purines in DNA following dimethylnitrosamine administration was studied in control and hypophysectomized rats. When given the same dose of this carcinogen (in mg/kg body weight) the formation of the major product 7-methylguanine and of the minor products 1-, 3- and 7-methyladenine and 3-methylguanine was slightly greater in the livers of hypophysectomized rats than in controls. The rate of loss of these products from the DNA was not affected by hypophysectomy. O6-Methylguanine levels were significantly greater in the hepatic DNA of hypophysectomized rats compared to controls after doses of dimethylnitrosamine ranging from 1 to 20 mg/kg. This difference was due to a slower rate of loss of this purine from the DNA in the hypothysectomized rats. Growth hormone treatment increased the rate of removal of O6-methylguanine in the hypophysectomized rats but did not restore the activity to that found in controls. The possible significance of these results in the induction of tumors by dimethylnitrosamine is discussed.


The moderately repetitive DNA sequences of Caenorhabditis elegans do not show short-period interspersion. In these studies we show that the moderately repetitive DNA sequences of Caenorhabditis elegans are not arranged in the characteristic short-period interspersion pattern of most eukaryotes. Rather, the moderately repetitive sequences are arranged in long arrays as in Drosophila and Apis. These findings indicate that this type of arrangement is more phylogenetically diverse and hence less exceptional than previously believed.


The influence of inhibitors on dimer removal and repair of single-strand breaks in normal and bromodeoxyuridine substituted DNA of HeLa cells. The elimination of cyclobutane pyrimidine dimers from the nuclear DNA of ultraviolet irradiated HeLa cells has been examined by means of chromatography and immunoautoradiography. The extent and duration of the process was similar when dimers were assayed by both methods, proving that the anti-sera recognized pyrimidine dimers. The rate of dimer excision did not differ through the cell cycle with the exception of mitosis during which no dimers were removed. Dimer excision is a relatively fast process which is terminated within a few hours, but it leaves many dimers in the DNA. Excision is depressed by inhibitors of semiconservative DNA synthesis that affect the DNA precursor pool or DNA polymerases. Cells whose DNA is partly substituted with bromodeoxyuridine instead of thymidine, repair single-strand breaks and remove dimers at the same rate but to different extents. On the other hand, inhibitors limit repair of breaks and removal of dimers to the same degree suggesting that the repair of the two types of lesion is coordinated.


Induction and repair of strand breaks and 3'-hydroxy terminals in the DNA of mouse brain following gamma irradiation. DNA was isolated from mouse brain after in vivo gamma-ray irradiation, treated with endonuclease S1 from Aspergillus oryzae if necessary, and analysed further by alkaline and neutral sucrose gradient centrifugation. In parallel, its template activity was determined by DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay as described previously. Similar experiments were performed with cultured mouse leukaemia cells (L5178Y) irradiated in vitro at 0 degrees C. Irradiation induced single- and double-strand breaks in the DNA of the brain with a yield of 1.0 and 0.1 break per 10(12) dalton per rad (100 eV/break and 770 eV/break), respectively. The yield of single-strand breaks in the brain was lower than that found in the cultured cells, whereas the yield of double-strand breaks was found to be almost the same in both cases. Treatment of irradiated DNA with single-strand-specific S1 endonuclease gave rise to further breaks detected on neutral sucrose gradient analysis. The yield of these breaks was also higher in the brain compared to the cultured cells. The increase per unit dose in the template activity of the DNA from the brain was found to be five times as much as that found in the cultured cells. Then, the average number of deoxyribonucleotides incorporated per break was determined on DNA which had experienced different treatments. The value for the brain DNA irradiated in vivo was found to be five times as much as that found for DNA treated with pancreatic deoxyribonuclease and 10 times as much as those found for DNA from the cultured cells and isolated brain nuclei irradiated in vitro at 0 degrees C. Thus, in vivo irradiation seemed to induce gaps with 3'-OH terminals in addition to simple breaks with or without 3'-OH terminals found in the cultured cells. Radiation-induced single-strand breaks and 3'-OH terminals in the DNA of the brain were repaired following irradiation. Approx. 20--40% of the terminals or breaks induced were, however, remaining at 3 h or more after irradiation, depending on the dose administered.


The rate of hydrolysis of methyl phosphotriesters in DNA under conditions used in alkaline sucrose gradients. Methyl phosphotriesters have been introduced into DNA, in vitro, by reaction with N-methyl-N-nitrosourea and the rate of degradation in alkali has been followed by measurements of the mean sedimentation coefficient using an analytical ultracentrifuge. In 0.3 M NaOH/0.7 M NaCl, a solution commonly used in alkaline sucrose gradient experiments, hydrolysis of the methyl phosphotriesters present is complete after 15 h at 20 degrees C, or after 2--3 h at 37 degrees C. In addition to breaks formed by the latter reaction there was a continuous background degradation of the DNA giving rise to 6.3 and 63 breaks/10(6) nucleotides per h at 20 degrees and 37 degrees C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.


Impairment of RNA synthesis and its recovery in angelicin photosensitized mammalian cells. A probe for DNA damage and repair. The template activity of DNA for ribosomal RNA transcription has been investigated in monkey kidney CV-1 cells after angelicin photosensitization in order to monitor the induction of lesions in the DNA and their possible subsequent disappearance, i.e. repair. Separate confluent cultures were submitted to a single angelicin treatment at different time intervals before incubation with [3H]uridine. The labeled RNA prepared from whole cells was analysed by polyacrylamide-agarose gel electrophoresis. The results indicate that: Angelicin monoadditions on DNA constitute transcription-terminating lesions which depress overall RNA synthesis, give rise to shortened RNA chains and modify the expression of transcriptional linked genes. CV-1 cells are able to repair, at least partially, the induced transcription-terminating lesions and progressively recover RNA synthesis with a reversion of the initially observed modifications. The repair seems to be independent of semiconservative DNA synthesis since fluorodeoxyuridine does not affect the recovery of RNA transcription. The present work also confirms the arrangement of rRNA genes in tandem behind a common operator in the order 18--28 S as previously determined in the same cells by a radiological mapping technique and reinforces the potential applicability of transcription analysis to the study of repair processes operating on physically or chemically induced damage in DNA.


Composition and properties of messenger ribonucleoprotein fragments containing and lacking polyadenylate. Ribonucleoprotein fragments were isolated from Ehrlich ascites cell messenger ribonucleoprotein (mRNP), immobilized on oligo(dT)-cellulose, by successive RNAase and formamide treatments and their RNA and protein moieties were characterized. A 8--10 S formamide-eluted (F) RNP primarily contained the poly(A) segment as an RNA moiety, as evidenced by its characteristic electrophoretic mobility, resistance to RNAase digestion, affinity for nitrocellulose filters and poly(U), and base composition. In contrast, the RNAase-eluted (R) fraction was composed of a 2--3 S RNP carrying a much shorter polyribonucleotide fragment of heterogeneous base composition and properties unlike poly(A). Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the protein moieties of the R fraction were composed primarily of 56 000, 67 000, and 71 000 molecular weight polypeptides whereas those of the F fraction contained the latter three polypeptides as well as a major species exhibiting a molecular weight of 81 000. The data demonstrate that RNA sequences other than poly(A) interact with protein in mRNP, that three major mRNP polypeptides are associated with both poly(A) and non-poly(A) RNA binding sites, and that one major polypeptide is specifically bound to the poly-(A) region of mRNP.


Size analysis of heterogeneous nuclear RNA in an aqueous gel system. We have analyzed the size distribution of heterogeneous nuclear RNA (hnRNA) from cultured murine cells in an aqueous gel system consisting of 1.8% polyacrylamide and 0.5% agarose. hnRNA, labeled in vivo with 32P at a low specific activity, was labeled in vitro at its 3'-end with NaB3H4, and fractionated by gel electrophoresis. The ratio of log 32P/3H cpm was determined for each gel fraction and shown to be linearly related to distance migrated. The ratio of 32P/3H cpm was used to predict the molecular weight of 28-S and 18-S RNAs and shown to be within approx. 10% of the known molecular weight. Denaturation of hnRNA with heat or dimethyl sulfoxide gave similar number and weight average molecular weight when analyzed by gel electrophoresis. The same hnRNA analyzed by sucrose gradient velocity sedimentation had a similar number average molecular weight but much higher weight average molecular weight.


Influence of toyocamycin on the assembly and processing of preribosomal ribonucleoproteins in the nucleolus of mammalian cells. The adenosine analogue toyocamycin inhibits the maturation of ribosomal RNA, but permits the synthesis of other RNA species, including 45 S preribosomal RNA. In this work, the dose vs. response analysis of rRNA processing upon toyocamycin treatment of L5178Y cells is studied. It is shown that the latter steps of rRNA processing are more affected than the earlier. The mechanism responsible for the lack of conversion of toyocamycin-containing 45 S RNA into mature rRNA has not yet been elucidated. In order to investigate whether protein factors are involved in this mechanism, the effects of toyocamycin on the ability of preribosomal RNA to bind proteins and on the assembly of nucleolar preribosomes are investigated. The analogue allows the formation and the accumulation, in the nucleolus, of ribonucleoprotein complexes which contain 45 S RNA and newly synthesized proteins, but cannot be converted to mature ribosomal subunits. These complexes are not clearly distinguishable from 80 S particles synthesized in the absence of toyocamycin, with respect to their sedimentation rate in linear sucrose gradients, to their protein/RNA ratio and to their density measured in metrizamide gradients.


Quantitative analysis of RNA, glycogen and nucleotides from different developmental stages of Ascaris lumbricoides (var. suum). Cold acid phenol extractions (Kirby, K.S. (1968) in Methods in Enzymology, Vol. XII B, pp. 87--99, Academic Press, New York) of homogenates from oocytes, zygotes and larval stages of Ascaris lumbricoides yield predominantly glycogen, some glycogen . phosphate complexes and guanine nucleotides, but only small amounts of RNA. Oocytes contain 10.0 pg of extractable 19 S and 26 S rRNA and 1.1 pg 4 S and 5 S RNA per cell. During the maturation of the oocyte to the zygote stage, the amount of 19 S and 26 S rRNA increases by 57%, and that of 4 S and 5 S RNA by 130%. Larval stages contain 62% more 19 S and 26 S rRNA, and 47% more 4 S and 5 S RNA than zygotes. The amount of the extractable glycogen decreases by about 80% and nucleotides by about 70% from oogenesis to the larval stage. The Kirby extract from homogenates of spermatids contains only traces of glycogen, glycogen . phosphate complexes and nucleotides, and 115 times less RNA per spermatid than oocytes. The nucleotide pool sizes of oocytes, zygotes and larval stages were determined; the pools consist of 71--88% guanine nucleotides. Spermatids contain only adenine nucleotides and an unidentified, nucleotide-like compound.


Changes in RNA polymerase activity in isolated mouse uterine nuclei during the decidual cell reaction. The artificially stimulated decidual cell reaction has been used as a model to study changes occurring in the uterus at the time of implantation. Activities of RNA polymerases I, II and III were measured in uterine nuclei isolated from ovariectomized non-primed mice, hormonally primed mice, and hormonally primed mice following stimulation of the decidual cell reaction. Activities of all three RNA polymerases increased following hormonal priming of ovariectomized mice. In nuclei from stimulated uterine horns, activities of RNA polymerases I and III increased 9 h after stimulation of the decidual cell reaction and remained elevated through 21 h. RNA polymerase II activity did not change following stimulation of the decidual cell reaction. These changes in RNA polymerase activities occur at the time of increased histone modifications and may result from changes in the template capacity.