Intramitochondrial positions of cytochrome haem groups determined by dipolar interactions with paramagnetic cations. E.p.r.(electron-paramagnetic-resonance) spectra of the ferricytochromes were studied in normal and 'nickel-plated' pigeon heart mitochondria and pigeon heart submitochondrial particles. NiCL2 added to either mitochondria or particles was bound completely to the membranes, but none was transported across the vesicles. Hence, any perturbations of the haem e.p.r. spectra by Ni(II) should occur only for those cytochromes in close proximity to the exterior surface. Whenever Ni(II) can approach to within 1 nm of cytochrome haem. the consequent acceleration of the haem e.p.r. relaxation kinetics should elicit dipolar line broadening. Relaxation acceleration should also increase the incident power level required to saturate the haem e.p.r. signal. In pigeon heart mitochondria, at least three e.p.r. resonances, attributable in part to cytochromes c1, bK and br, are observed at gz=3.3 resonance. In these submitochondrial particles, the peak at gz=3.5 is missing, and the resonance at gz=3.6 resolves into two components, neither of which is sensitive to added Ni(ii). Addition of free haemin (ferric, a paramagnetic anion) to intact mitochondria elicits the same e.p.r. signal changes as does a preparation of submitochondrial particles. Saturation curves for cytochrome oxidase obtained for e.p.r. spectra of the high-spin form (g = 6) and the low-spin form (gz=3.1) also reveal no effect of Ni(II) on the haem e.p.r. relaxation in either mitochondria or inverted submitochondrial particles. Further, Ni(II) fails to alter the spectra or saturation properties of cytochrome c in either mitochondria or submitochondrial particles therefrom. Only with a 50-fold molar excess of Ni(II) can one accelerate the e.p.r. relaxation of cytochrome c in aqueous solution, although other more subtle types of magnetic interactions may occur between the cytochrome and either Ni(II) or ferricyanide. Addition of haemin to mitochondria likewise failed to alter the e.p.r. characteristics of either cytochrome c or cytochrome oxidase. The present observations strongly suggest that cytochromes bK, br and c1 reside on the exterior surface of the inner mitochondrial membrane. On the other hand, we find no positive evidence for the location of cytochrome c or cytochrome oxidase haem groups within 1 nm of either membrane surface. Because of possible shielding effects from the protein moieties, however, we cannot unequivocally assign the location of the haem groups to the membrane interior. The present results are not inconsistent with the observations of other investigators who used different techniques. However, it is clear that any model of energy coupling in mitochondrial oxidative phosphorylation must account for the positioning of all the b-c cytochrome haem groups on the outside.


Intramitochondrial positions of ubiquinone and iron-sulphur centres determined by dipolar interactions with paramagnetic ions. E.p.r. (electron-paramagnetic-resonance) spectra of ubisemiquinone (QH) organic radicals and all of the known iron-sulphur centres were studied in normal and 'nickle-plated' pigeon heart mitochondria, submitochondrial particles and submitochondrial particles from which succinate dehydrogenase had been removed. Incubation of pigeon heart mitochondria, submitochondrial particles or succinate dehydrogenase-depleted submitochondrial particles with substrate in the presence of pure O2 results in the accumulation of Q-H. In mitochondria, the e.p.r. spectrum of Q-H is characterized by in-homogeneous line broadening. A heterogeneous population of semiquinones appears to be partly responsible for these effects in mitochondria. Additon of Ni(II) to mitochondria renders saturation of the Q-H resonance more difficult. On the other hand, the resonance in either submitochondrial particles or succinate dehydrogenase-depleted particles is narrower than the same spectrum in mitochondria, and saturates like a homogeneous line. The presence of Ni(II) in either of these preparations, further, has no effect on either the A-H spectrum or the saturation curve. Therefore QH appears to be situated on the exterior surface of the mitochondrion. Likewise, the e.p.r. spectra and saturation curves of iron-sulphur centre N-2 exhibit characteristics of inhomogeneous line broadening, not only in intact mitochondria but also in both submitochondrial particles and succinate dehydrogenase-depleted particles. Because of the small pool size of centre N-2, this effect is likely to arise from a spin interaction with some other component in the membrane. Ni(II) has no effect on the saturation in centre N-2 in mitochondria or submitochondrial particles, and only a marginal effect in the succinate dehydrogenase-depleted preparation. These results are indeterminate with respect to the position of centre N-2 in the membrane; but suggest that its distance from the succinate dehydrogenase binding site is on the order of 1 nm. All of the other ferredoxin-type iron-sulphur centres in both preparations were not affected by paramagnetic ions. Homogeneous e.p.r. spectra and saturation curves are observed for both of the HiPIP-type (high-potential iron-sulphur protein-type) iron-sulphur centres in mitochondrial centres S-3 and bc-3. Addition of No(II) to intact mitochondria results in a dipolar interaction with centre bc-3. No effect was observed on centre S-3 in either preparation. A comprehensive model is presented for the structure of the respiratory electron-transport system in mitochondria, based on e.p.r. relaxation studies in the present and the preceding paper. There is no direct evidence for transmembrane electron flow through any of the known energy-coupling sites in mitochondria, so that direct hydrogen atom transfer across the membrane (as a combination of H+ translocation coupled to electron flow) does not occur...


Hypertriglyceridemia in the diabetic rat. Defective removal of serum very low density. Streptozotocin-induced diabetic rats show a marked fasting hypertriglyceridemia. It appears that only the very low density lipoprotein (VLDL) fraction is increased. VLDL from either normal or diabetic rats was labelled in vivo in the triglyceride moiety with [3H]palmitate and isolated. Both preparations, if injected intravenously into recipient rats, are removed more slowly from the circulation of diabetic rats as compared to normal rats, resulting in a reduction of the fractional catabolic rate (F.C.R.) by 70%. However, the absolute catabolic rate (turnover) of VLDL triglycerides was not changed in diabetics. It is concluded that the hypertriglyceridemia of the the diabetic rat is caused by a defective removal mechanism of VLDL triglycerides. The F.C.R. of 125I-labelled low density lipoproteins and high density lipoproteins is only 1-3% of the value for VLDL triglycerides and unchanged in diabetic rats.


Intravenous fat tolerance. Correlation with very low density lipoprotein apoprotein B kinetics in man. The intravenous fat tolerance test (IVFTT) has been introduced as a measure of the fractional catabolic rate of the endogenous triglyceride of plasma. To assess the validity of this test we have compared this test in 21 normal and hyperlipidaemic subjects with direct measurement of the fractional catabolic rate of autologous radio-iodinated VLDL. There was a strong positive correlation between the rate constant K2 of the IVFTT and the fractional catabolic rate of VLDL-apolipoprotein B (r=+0.87). The two rates were considerably different in magnitude. The IVFTT appears to be a valid index of the fractional catabolic rate of VLDL. Its limitations and uses are discussed.


Effect of phospholipids on cholesterol-induced modifications in mouse brain. Mice on an atherogenic diet for 40 days show a decrease in brain content of catecholamines, cyclic AMP and in dopamine degradation, and modification of the glycolytic pathway. The metabolic changes are paralleled by changes in behaviour, i.e. decrease in spontaneous motor activity and in conditioning avoidance response. The decrease in dopamine degradation and in behaviour parameters is partly due to the propylthiouracil present in the diet. Endovenous treatment with sonicated dispersions of bovine brain phospholipids induces a modification in the parameters of behaviour and metabolism. The possibility is discussed that some of the defects arising during the atherogenic diet are related with the establishment of a hypoxic state.


Metaformin: an antiatherosclerotic agent modifying very low density lipoproteins in rabbits. The composition of very low density lipoproteins (VLDL: d less than 1.019) of New Zealand male rabbits reciving cholesterol (2 g/day) and metformin (135 mg/kg/day) is investigated. These rabbits, while showing only a slight reduction of plasma cholesterol levels, as compared to cholesterol-fed (h.c.) animals, show a marked decrease of the aortic cholesterol esters and atheromatous process. VLDL from the cholesterol + metformin group (h.c. + met), as compared to the h.c. animals, are homogenous in size and not separable into VLDL-1 and VLDL-2 subfractions by Sepharose 4B chromatography. These findings are confirmed by electron microscopy, which shows homogeneity of particle size, as well a decreased tendency of h.c. + met VLDL to aggregate. Chemical composition of h.c. + met VLDL is characterized by increased triglycerides and phospholipids, while the percentage of cholesterol esters is not significantly decreased. Phospholipid distribution of h.c. + met VLDL shows a significant decrease of sphingomyelin and increased phosphatidylinositol, the latter both as compared to h.c. and control VLDL. Apoprotein pattern of h.c. + met VLDL in polyacrylamide gels shows a relative increase of peptides with C mobility and a decrease of proteins corresponding to the arg-rich peptides. These findings exemplify a case of altered lipoprotein composition and decreased atheromatosis, in the presence of marked hypercholesteremia.


[Researches on the water quality of the river Po and its tributaries between Cremona and Casalmaggiore. I. materials and methods (author's transl)]. During 1971 a research program about the status of pollution of the river Po and its tributaries was started by six Institutes of Hygiene altogether (Turin, Pavia, Milan, Parma, Modena and Ferrara Universities) with the Institute for Water Research of C.N.R. and Hydrographic Office of Magistracy of the Po. The role of the Parma University Hygiene Institute was to study the water qualities of the river Po between Cremona and Casalmaggiore and of two right side tributaries, Arda-Ongina and Taro. The hydrogeographical characteristics of the examined reach are reported in the present note, with special reference to the hydrological conditions, basin area, inhabitants and industrial typology. The sampling stations were placed upstream and downstream Cremona town, near Casalmaggiore and at the mouth of Arda-Ongina and Taro. Moreover the methods, for chemical and bacteriological analysis are reported.


High plasma cholesterol in mink (Mustela vison) without atherosclerosis. Mink fed a commercial ration moderately high in cholesterol or fed a cholesterol-free semipurified diet have plasma cholesterol-free semipurified diet have plasma cholesterol concentrations similar to that found in human beings living in industrialized countries. In contrast with human beings, 80% of the plasma cholesterol in mink is carried in the high density lipoprotein fraction. Aortas and coronary arteries from animals up to 8 yr old were found to be free of fatty streaks and atherosclerotic plaques, both grossly and microscopically.


Metabolic effects of carbon monoxide in relation to atherogenesis. The acute and chronic effects of carbon monoxide (CO) are reviewed. Direct effects of CO-induced hypoxia on arterial metabolism may facilitate deposition of cholesterol in the intimal surface and exacerbate existing atherosclerosis. Other studies indicate that CO may alter hepatic metabolism so as to inhibit both the secretion of very low density lipoproteins and the clearance of chylomicron remnants. The former change would diminish and the latter enhance atherogenesis. These findings are considered and avenues of future investigation suggested.


Chemical composition of ultracentrifugal fractions in different patterns of human atheroslcerosis. The chemical composition of ultracentrifugal fractions of VLDL (d less than 1006), LDL (d 1006-1063) and HDL (d less than 1063) has been studied in males affected by atherosclerosis of different vascular beds. Thirty-seven subjects affected by post-infarction cardiopathy (M.I.) showed significantly higher values of total-C, VLDL-C and LDL-C when compared to 52 controls. Twenty-three patients affected by non-occlusive ischaemic heart disease (I.H.D.) showed higher values than controls of total-C, VLCL-C, LDL-C, total TG, VLDL-TG, and GDL-TG. Twenty-three patients with atherosclerosis of the inferior limbs (P.A.) were characterized by increased levels of total-TG, VLDL-TG, VLDL-C, HDL-C. A group of patients who had suffered a stroke from cerebro-vascular disease (C.V.D.) did not show any significant difference from controls. In the M.I. group, 56% of the patients had a high level of C-VLDL. Patients with I.H.D. were characterized mostly by an increase in C-LDL, Patients with P.A. showed the highest values of total -TG, VLDL-TG and LDL-TG. Some of the observed differences are probably due to different metabolic backgrounds. Some other differences may be due to variations in dietary habits after heart infarction. Patients with levels of plasma cholesterol and triglyceride beyond the 90th percentile of the normal group showed many abnormalities in the chemical composition of their lipoproteins. It is noteworthy that increased amounts of cholesterol may collect in lipoprotein classes different from LDL while increased amounts of triglyceride may collect in classes different from VLDL.


Lecithin: cholesterol acyl transfer rate in plasma and its relation to lipid and lipoprotein concentrations in primary hyperlipidemia. Plasma lecithin: cholesterol acyl transfer (LCAT) rate and concentrations of lipids in plasma and lipoproteins were studied in 107 hyperlipidemic subjects. In all types of hyperlipoproteinemia LCAT rates were higher than in a normolipidemic reference group. LCAT rates were highest in type IV and V. There was a considerable overlap of LCAT rates between type IIa, IIb and reference subjects. The LCAT rate correlated positively with very low density lipoprotein concentration, body mass and excess body mass. Low density lipoprotein concentration correlated positively with the LCAT rate only in the reference group. The high density lipoprotein concentration correlated negatively with the LCAT rate. It was suggested that the LCAT rate in vitro reflects the in vivo turnover of cholesteryl esters as a part of the turnover of apoprotein-B containing lipoprotein complexes in plasma. The results might then indicate an inflow rate of lipoproteins in plasma that is increased in most type IV cases but normal or only moderately increased in type IIa and IIb subjects. Analysing the relations between the LCAT rate and the concentrations of lipids in plasma by multiple regression indicated hypothetically deficiencies of lipoprotein removal from plasma in half of type IIa and one third of type IIb subjects.


Administration of antigen by intralymphatic route in the rat. II. Results in tumor active immunotherapy (delta). Preliminary report. Tumor growth and survival time were followed in rats transplanted with Morris hepatoma 5123 and previously injected with a crude homogenate of the same tumor by the intradermic or intralymphatic route. Female rats treated by the intralymphatic route showed a longer survival time and impaired tumor growth. Male rats did not show any difference.


Virus-like antigen associated with hepatitis A: investigations in children's acute hepatitis. Stool specimens, collected from 8 children with viral hepatitis [5 of type A, presumably, and 3 of type B (HBsAg-positive)] during the acute phase, were examined in the electron microscope. The presence of 27-nm virus-like particles, aggregated by the convalescent serum of a chimpanzee infected with MS-1 and not by the serum of the same animal drawn before infection, was detected by immune electron microscopy (IEM) in the stools of 3 out of the 5 children with suspected hepatitis A. The virus-like particles (HA-Ag) present in the stools were aggregated by the serum drawn from the corresponding subject during convalescence and not by that obtained in the acute phase of the disease. Employing as antigen a stool particle preparation purified by isopycnic banding in cesium cloride, it was pointed out that, in all 5 children with suspected hepatitis A, antibodies capable to cause the aggregation of HA-Ag particles, appeared. Anti-HA antibodies turned out to be present also in three lots of commercial immunoglobulins. In the three subjects with type B hepatitis (HBsAg-positive) neither virus-like particles with a 27-nm diameter were observed, nor anti-HA antibodies appeared in the convalescent serum.


Sero-epidemiological survey on human and swine influenza A. Situation at the onset of summer 1976. A serological survey was performed to detect the presence of HI antibodies against the swine-like A/New Jersey/76 (Hsw1 N1) and the human A/Victoria 3/75 (H3N2) virus strains in the sera of 700 subjects of different age, and of 244 swine. HI antibodies against A/New Jersey/76 strain were not detected in 308 subjects younger than 31 years. They turned out to be present in 8% of the subjects from 31 to 43 years, always at low titre, and at a different titre, in 50% of the subjects from 44 to 58 years and in 90% of the subjects older than 58 years. None of the 244 swine tested from breeding farms of Liguria, Piedmont and Lombardy turned out to be endowed with antibodies against A/New Jersey/76 virus. This shows that the swine-like virus, which caused infections in the human population up to about 1930, did not circulate any more subsequently and that the swine also are now free from this infection. As far as the A/Victoria 3/75 strain is concerned, HI antibodies were detected in the different age groups with a frequency ranging between 41 and 69%. The fact that in many subjects antibodies were present at low titre suggests that their presence should be only partially brought back to infections caused by A/Victoria 3/75 but that in many cases it is the expression of a heterologous response to previous infections caused by other A H3N2 strains. HI antibodies against A/Victoria 3/75 were found also in 13% of the swine examined. A group of older people, who had been regularly immunized for years with the inactivated (A H3N2-B vaccine) showed an HI antibody titre significantly higher in comparison with subjects of equivalent age not vaccinated both against A/Victoria 3/75 and A/New Jersey/76.


[Liver resection in children. Experience in 10 cases]. The authors report their experience with ten major hepatic resections; six on the right lobe and four on the left. Six of the resected masses were hepatoblastomas, two were hamartomas, was a hemangioma, and the last one a pheochromocytoma of the right adrenal gland with invasion to the liver next from the affected site. The four patients with major hepatic resection of the left lobe are at present alive, ranging from two months to two years after surgery. Of the six patients with major resection of the right lobe, three died during surgery, another one died late post-operatively and one died a year after surgery from generalized metastasis of an hepatoblastoma. The sixth patient is alive with no complications, four months after surgery. The authors used two types of hepatic resections and they make some recommendations for achieving better results in this type of operation.


Ultrastructure of abnormal membrane inclusions in nuclei of human myocardial cells. The ultrastructure of the nuclear envelope was studied in cardiac muscle cells of 12 patients undergoing cardiopulmonary bypass surgery. Two main types of membrane inclusions, globular and tubular, were observed in the nuclei. The globular type was found in 6 patients. The globular inclusions were about 0-5mu to 1-5mu in diameter and lined by two unit membranes equal in structure to that of the nuclear envelope. The lining was probably always in continuity with the nuclear membranes. Such inclusions contained a granular matrix in which no cytoplasmic organelles could be seen, with the exception of a limited number of ribosomes. The tubular type of nuclear inclusion was observed in the cardiac tissue of 3 patients. These tubules ran in straight or slightly bent segments of about 0-2 mu to 1-6 mu length, interrupted by bendings at obtuse or right angles. The diameter of their lumina measured about 300 A to 800 A. Such tubules were nearly always found in areas of the nucleoplasm rich in heterochromatin. Nuclear pore-like structures were occasionally found in the tubular wall. The tubular wall consisted of a single unit membrane shown to be in continuity with the inner membrane of the nuclear envelope. Since these nuclear aberrances have been observed at all of the stages examined, the possibility is considered that they may represent a specific nuclear response to the process of cardiac hypertrophy.


Effect of pretreatment with immune serum on murine sarcoma virus (Moloney) tumour induction and growth. Regressor serum from MSV-M-infected mice markedly reduced MSV-M oncogenesis when administered i.p. (0-1 ml/mouse) as much as 30 days before i.m. MSV-M infection in adult BALB/c mice. The regressor serum activity appeared to be directly dependent on the amount of IgG, as shown by: (1) inactivity of sera which have low virus-neutralizing antibody content; (2) high effectiveness only of the IgG serum fraction; (3) inactivity of regressor serum incubated with anti-mouse gamma-globulin serum. The regressor serum activity was specific and could not be ascribed to interferon or interferon-inducing factors, antigen-antibody complexes or free antigen. The activity was not suppressed by sublethal irradiation (380 rad) of recipient mice. These results suggest that the activity of regressor serum administered before MSV-M infection is mediated through sensitization of host cells which are not radiosensitive.


Lung cancer in Hong Kong Chinese: mortality and histological types, 1960-1972. Age-adjusted mortality from lung cancer rose rapidly in both males and females in Hong Kong from 1960-1972. The relative frequency of epidermoid carcinoma increased in male bronchial biopsies but not in lung biopsies, resections, or autopsies; there was a decline in small-cell anaplastic carcinoma. In both males and females the ratio of Kreyberg Group I (epidermoid and small-cell anaplastic) to Group II (adenocarcinoma and carcinoid) tumours did not increase, despite an 80% rise in mortality from lung cancer. Adenocarcinoma was the most common type in females, despite the high mortality from lung cancer. It is speculated that cigarette smoking might produce a different pattern of histological types among Hong Kong Chinese, or that additional aetiological factors may be operating there.


Histochemical and histological effects of lead on the liver and kidney of the dog. In a series of 3 experiments, beagle dogs were dosed orally with lead carbonate and the histochemical and histological changes in the liver and kidney assessed. Dosing at 50 mg/kg per day for 5 weeks resulted in well documented histological changes in the kidney and hydropic degeneration in the liver; significant alterations in the activity of the majority of enzymes studied were also seen in both organs. In dogs dosed for one week at 50 or 100 mg/kg no histological changes were seen and histochemical alterations were mainly confined to the dehydrogenases and NADPH diaphorase. A third group of dogs were dosed for 3 weeks; during a subsequent recovery period of almost 2 months the mild clinical effects produced by lead during the dosing period were quickly reversible except in 2 dogs. At the end of the recovery period histochemical alterations were evident in both organs of these 2 dogs principally shown by a reduction in the dehydrogenases of the liver. The findings are interpreted as an effect by lead on a range of cellular enzymes particularly those involved in energy production, these effects being still demonstrable after an extended recovery period.


Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli. The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.


Fluorescence and nucleotide binding properties of Escherichia coli uridine diphosphate galactose 4-epimerase: support for a model for nonstereospedific action. The fluorescence emission spectrum for reduced diphosphopyridine nucleotide (DPNH) in Escherichia coli uridine diphosphate galactose 4-epimerase-DPNH complexes has a maximum at 435 nm, which is about twice as intense when the excitation is at 280 nm as at 340 nm. The fluorescence excitation spectrum monitored at 460 nm has two maxima, one at 340-345 nm and another about twice as intense at 280 nm. The polarization of DPNH fluorescence by these complexes is 0.43-0.44 compared with 0.46 for DPNH immobilized in propylene glycol at -20 degrees C. The small degree of fluorescence depolarization is due to rotational relaxation of the protein, relaxation time 205 ns. The excited-state lifetimes in epimerase-DPNH-nucleotide complexes are 3.5-4.2 ns. The fluorescence data show that the dihydropyridine ring in these complexes is highly immobilized and exhibits no detectable independent motion relative to rotational motions of the protein. The inhibition constants for uridine monophosphate (UMP) and 2,2,6,6-tetramethyl-4-piperidinyl-1-oxyl uridyl pyrophosphate acting as competitive reversible inhibitors of epimerase-DPN+ are 1.2 and 0.2 mM, respectively, at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5. A collection of Ki and Km values for uridine nucleotide inhibitors and substrates indicates that the principle substrate binding interactions involve the nucleotide moieties of substrates. Dissociation constants for uridine nucleotides dissociating from epimerase-DPNH-nucleotide complexes, measured by ultraviolet absorption and fluorescence techniques, are 12 muM for UMP, 14 muM for UDP-hexopyranoses, 4 muM for UDP-pentopyranoses, 27 muM for p-bromoacetamidophenyl uridyl pyrophosphate, 0.14 muM for UDP-4-ketohexopyranose intermediate, and 0.36 muM for UDP-4-ketopentopyranose intermediate at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5. Analysis of these data shows conclusively that the major part of the binding free energy for UDP-4-ketopyranose intermediates binding to epimerase-DPNH is attributable to the uridylpyrophosphoryl components and that the glycosyl-binding free energies are much smaller. The data show that the action of this enzyme does not require tight binding between the active site and glycosyl groups of either substrates or intermediates, although there is favorable binding of the uridylpyrophosphoryl components, particularly by epimerase-DPNH. It is postulated that nonstereospecific action results from and depends upon relatively weak, nonspecific active site binding of glycosyl groups in substrates and intermediates and that the uridylpyrophosphoryl groups serve as binding anchors in the epimerization process.


A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label. The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.


Interaction of detergents with cytochrome c oxidase. The binding of ionic and nonionic, nondenaturing detergents to cytochrome c oxidase has been examined. All bind and displace part but not all of the phospholipid that is associated with the enzyme after isolation. From 6 to 10 phospholipid molecules, depending on the detergent used, do not exchange and these are mostly diphosphatidylglycerol molecules as first shown by Awasthi et al. ((1971) Biochim. Biophys. Acta 226, 42). The binding of Triton X-100 and deoxycholate to the cytochrome c oxidase complex has been studied in detail. Both bind to the enzyme above their critical micelle concentrations: Triton X-100 in the amount of 180 +/- 10 molecules per complex and deoxycholate in the amount of 80 +/- 4 molecules per complex. In nonionic detergents, cytochrome c oxidase exists as a dimer (4 heme complex). The enzyme is dissociated into the monomer or heme aa3 complex by delipidation in bile salts. Activity measurements in different detergents suggest that cytochrome c oxidase requires a flexible, hydrophobic environment for maximal activity and that the dimer or 4 heme complex may be the active species.


Thermodynamic limits on the size and size distribution of nucleic acids synthesized in vitro: the role of pyrophosphate hydrolysis. The free-energy change of phosphodiester bond formation from nucleoside triphosphates is more favorable than with nucleoside diphosphates as substrates. Base-stacking interactions can make significant contributions to both delta G degrees ' values. Pyrophosphate hydrolysis when it accompanies the former reaction dominates all thermodynamic considerations. Three experimental situations are discussed in which high-molecular-weight polynucleotides are synthesized without a strong driving force for covalent bond formation. For one of these, a kinetic scheme is presented which encompasses an early narrow Poisson distribution of chain lengths with ultimate passage to a disperse equilibrium population of chain sizes. Hydrolytic removal of pyrophosphate expands the time scale for this undesirable process by a factor of 10(9), while it enormously elevates the thermodynamic ceiling for the average degrees of polymerization in the other two examples. The electron micrographically revealed broad size population from an early study of partial replication of a T7 DNA template is found to adhere (fortuitously) to a disperse most probable representation. Some possible origins are examined for the branched structures in this product, as well as in a later investigation of replication of this nucleic acid. The achievement of both very high molecular weights and sharply peaked size distributions in polynucleotides synthesized in vitro will require coupling to inorganic pyrophosphatase action as in vivo.


Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits. Following ribonuclease digestion of methyl-3H-labeled B77 avian sarcoma virus RNA subunits, methylated oligonucleotides were isolated by diethylaminoethylcellulose chromotogrpahy. Partial nucleotide sequences were deduced from the known enzymatic specificities of the ribonucleases. In addition to methylated nucleosides in the 5'-terminal cap structure, m7G(5')GmpCp, N6-methyladenosine(m6A) was found to be present in only two internal sequences of the RNA molecule, Gpm6ApC and Apm6ApC. The average numbers of methylated nucleosides per RNA subunit are about 12-13 in Gpm6ApC, 1-2 in Apm6ApC, and 2 in m7GpppGmpCp. The sequences containing m6A in B77 sarcoma virus RNA are identical to m6A-containing sequences previously reported for the bulk mRNA from HeLa cells (Wei, C.M., Gershowitz, A., and Moss, B. (1976), Biochemistry 15, 397-401). Analysis of the oligonucleotides produced by RNase A digestion indicated that the sequence of bases on the 5' side of these trinucleotides is not specific. The oligonucleotide profile, however, was highly reproducible in different virus preparations. This suggests that the methylations occur at specific positions on the RNA molecule. Some of the methylated oligonucleotides produced by RNase A digestion appear to be present in less than molar amounts. Several hypotheses are proposed to explain this result.


Characterization of DNA strand breakage in vitro by the antitumor protein neocarzinostatin. The antitumor protein antibiotic neocarzinostatin causes strand scission of DNA in vitro in the presence of a sulfhydryl compound. The breaks are single stranded in nature and bear 5'-phosphoryl termini. All four deoxymononucleotides are recoverable at the 5'-ends of the cleavage sites although a higher proportion of dGMP and TMP are consistently found. The lesions are not repairable with polynucleotide ligase from Escherichia coli. A quantitative assay was developed to determine the pH profile and time course of the reaction. Data from protection experiments with synthetic and natural DNAs indicate the requirement for thymidylic acid and deoxyadenylic acid in the DNA for cutting. In DNA-RNA hybrids, riboadenylic acid can substitute for deoxyadenylic acid, whereas ribouridylic acid cannot substitute for thymidylic acid. Release of thymine is detected, and the amount of release correlates well with the number of strand scissions.


Synthesis and template-directed polymerization of adenylyl(3'-5')adenosine cyclic 2', 3'-phosphate. Adenylyl(3'-5')adenosine cyclic 2',3'-phosphate (A-A greater than p) was synthesized and its polymerization was attempted under various conditions inthe presence of poly(uridylic acid) and1,3-propanediamine. Reaction at -20 degrees C for 16 days gave polymerized products (up to the 8-mer) in 15% yield and was proved to be dependent on the template. Reaction at 0 degrees C for 16 days gave more extensive (up to the 10-mer) and more efficient (35%) polymerization. The newly formed phosphodiester linkage was exclusively 2'-5'. These results are discussed in comparison with the monomer-condensation reaction.


Poly(adenosine diphosphate-ribose) polymerase: the distribution of a chromosome-associated enzyme within the chromatin substructure. The distribution of a chromatin-bound, nuclear protein modifying enzyme, poly (adenosine diphosphate-ribose) polymerase, and its product, poly(ADP-ribose), among various fractions of sheared and nuclease-digested HeLa cell chromatin has been examined. Epichlorohydrin-tris(hydroxymethyl)aminomethane-cellulose and glycerol gradient fractionation of solubilized chromatin indicated that poly(ADP-ribose)polymerase activity was associated primarily with the template active regions (euchromatin), whereas the transcriptionally inert chromatin fractions were found to contain relatively low levels of ADP-ribosylating activity. When isolated HeLa cell nuclei were digested in situ with micrococcal nuclease and the resultant chromatin was fractionated into nucleosome monomers (v bodies) and oligomers by sucrose gradient centrifugation, only material sedimenting faster than the 11S monomers was found to contain appreciable poly(ADP-ribose) polymerase activity. If, on the other hand, isolated HeLa cell nuclei were first incubated with labeled NAD, the substrate for poly(ADP-ribose) polymerase, prior to the preparation and fractionation of nuclease-digested chromatin, it was found that those chromatin fractions which possess significant poly(ADP-ribose) polymerase activity (nucleosome oligomers) are relatively deficient in the labeled product of this enzyme, and that a considerable portion of the homopolymeric product is ultimately associated with the 11S v bodies. Additional evidence is presented which indicates that the absence of nucleosome monomer-associated poly(ADP-ribose) polymerase activity is not due to the absence of a suitable acceptor on these structures, and that the activity of this enzyme within the chromatin is most probably dependent upon the physical integrity of the oligomeric structures themselves.


Different metal-binding properties of the two sites of human transferrin. Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity.


Evidence that the catalytic and regulatory functions of carbamylphosphate synthetase from Escherichia coli are not dependent on oligomer formation. Carbamyl-phosphate synthetase from Escherichia coli is an allosteric enzyme which undergoes reversible association reactions in phosphate buffer. The positive allosteric effectors, ornithine, inosine 5'-monophosphate (IMP), and ammonia, facilitate oligomer formation, whereas uridine 5'-monophosphate (UMP), a negative effector, prevents or decreases oligomer formation. When the enzyme is immobilized by reaction with activated Sepharose, under conditions where the enzyme exists only as a monomer, nearly full catalytic activity is retained and the effects of ornithine, IMP, and UMP on the catalytic activity as a function of MgATP concentration are not significantly altered. Gel-filtration chromatography on Sephadex G-200 of catalytic quantities of the enzyme in the presence of all substrates showed that the elution volume was the same as that measured for the enzyme under conditions where it is known to exist in the monomer form. The specific activity of the enzyme does not increase when the concentration of the enzyme is increased 100-fold from a concentration at which the enzyme exists as monomer to a level at which the enzyme exists predominantly as oligomer. These results indicate that the monomer form of the enzyme is the principle active species and that oligomer formation is not directly related to enzyme activity or enzyme regulation.


Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli. The binding of ornithine and inosine 5'-monophosphate (IMP), positive allosteric effectors, and of uridine 5'-monophosphate (UMP), a negative allosteric effector, to carbamyl-phosphate synthetase from Escherichia coli was studied by the technique of equilibrium dialysis. The monomeric form of the enzyme has one binding site for each of the three allosteric ligands. The binding of UMP is inhibited by ornithine, IMP, MgATP, and ammonia (also a positive allosteric effector). Bicarbonate, L-glutamine, and adenosine 5'-triphosphate (ATP) (Mg2+ absent) had no effect on the binding of UMP. The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer. The binding of ornithine was inhibited by UMP and ammonia, enhanced by MgATP, MgADP, and IMP, and not affected by bicarbonate, L-glutamine, or ATP (Mg2+ absent). Ornithine and ammonia probably bind to the same site on the enzyme. The binding of IMP is facilitated by ornithine and ammonia, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site. The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing one or the other of two different conformational states of the enzyme which are in equilibrium with each other (Anderson, P.M., and Marvin, S.V. (1970), Biochemistry 9, 171). According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.


Effect of specific trifluoroacetylation of individual cytochrome c lysines on the reaction with cytochrome oxidase. We have prepared three different cytochrome c derivatives, each containing a single specifically trifluoroacetylated lysine at residues 13, 55, and 99, respectively. The only modification that affected cytochrome c oxidase (EC 1.9.3.1) activity was that of lysine-13 at the top of the heme crevice. Trifluoroacetylation of lysine-13 increased the apparent Michaelis constant fivefold compared to that of native cytochrome c, but did not affect the maximum velocity. Trifluoroacetylation of lysine-55 at the left side of the cytochrome c molecule did not affect cytochrome oxidase activity in any way, nor did trifluoroacetylation of lysine-99 at the rear of the cytochrome c molecule. This indicates that the cytochrome oxidase binding site on cytochrome c involved only the front of the cytochrome c molecule and those lysines immediately surrounding the heme crevice.


Spin-label studies on the aqueous regions of phospholipid multilayers. Water-soluble spin labels were used to study dimyristoyllecithin (DML) phospholipid multilayers. Previous studies report that there is a "bound" water region associated with dimyristoyllecithin containing about 10 molecules of water per phospholipid, a "trapped" water region located between the lamellae containing approximately 11 molecules per phospholipid, and a "ftion show that certain water-soluble spin-label mol-cules have their motional properties differentially modified by these three water environements. Furthermore, the labels also reveal the onset of lipid-phase transitions even though they have high water solubility. A phosphate-containing spin label demonstrated strong an isotropic motion in the lipid-water system above the phase transition but not below. The addition of cholesterol to the DML-water system removed the anisotropic motion of 2,2,6,6-tetramehtyl-4-phosphopiperidine-N-oxyl (Tempophosphate) and obscured the detection bound, trapped, and free water. In addition to the change-charge interactions between Tempophosphate and DML, two other spin labels were used both in the charged and uncharged states. 2,2,6,6-Tetramethyl-4-aminopiperidine-N-oxyl (Tempamine) in the charged state showed extremely strong anisotropic motion, presumably due to the interaction between the charged amine and the phosphate group of DML. When only partially charged, Tempamine showed much less anisotropic motion. PCA was analyzed at pH values where the carboxyl group was protonated and unprotonated. The resulting interaction was different at the two pH values. These water-soluble spin labels mimic ionic or nonionic solutes. Upon freezing, the spin labels are shown to be expelled from the ice regions into the remaining aqueous regions. The usefulness of this approach in studying solute behavior when freezing occurs and potential studies involving aqueous regions of cytoplasm are considered.


Actinomycin D complexes with oligonucleotides as models for the binding of the drug to DNA. Paramagnetic induced relaxation experiments on drug-nucleic acid complexes. Mn(II) ions have been used as a paramagnetic probe to investigate the geometry of drug-oligonucleotide complexes. Nuclear magnetic resonance and electron spin resonance experiments show that Mn(II) ions bind approximately two orders of magnitude stronger to the 5'-terminal phosphate group than to the 3'-5' phosphodiester linkage of deoxydinucleotides. By using mixtures of nucleotides in which only one nucleotide contains a terminal phosphate group, the location of the Mn(II) ion in the drug-nucleotide-Mn(II) complexes may be preselected. The paramagnetic induced relaxation of the nuclear spin systems in these complexes has been used to investigate the geometry of these complexes. These data confirm that actinomycin D is able to recognize and preferentially bind guanine (as opposed to adenine) nucleotides in the quinoid portion of the phenoxazone ring, while both adenine and guanine will bind to the benzenoid portion of the phenoxazone ring. These results suggest that stacking forces are primarily responsible for the general requirement of a guanine base when actinomycin D binds to DNA.


bc1-Complex from beef heart. One-step purification by hydroxyapatite chromatography in Triton X-100, polypeptide pattern and respiratory chain characteristics. A new simple method for the purification of the bc1-complex has been developed. The polypeptide composition of the complex was analysed by dodecyl sulfate-polyacrylamide gel electrophoresis. The content of chain components and phospholipids was determined. The b-type cytochromes were further characterized by their absorbance spectra and midpoint potentials. (1) Starting from a Triton X-100 extract of submitochondrial particles supplemented with antimycin, the bc1-complex is purified by adsorption chromatography on hydroxyapatite with citrate as specific eluant. (2) The complex splits in dodecyl sulfate into five main polypeptides with apparent molecular weight of 47, 44, 31, 11 and less than 10 kdalton. (3) The  purified complex has a heme-b content of 8.0 mumol/g protein and a cytochrome c1 content of 3.8 mumol/g protein. (4) The cytochromes show the typical absorbance spectra of cytochromes b-562 and b-565 and are present in approximately equal amounts with midpoint potentials of Em7 = + 100 mV and Em7 = + mV respectively. Carbon monoxide does not bind to the cytochromes. (5) The nonheme iron protein content of the complex is diminished to 0.6 mumol/g protein. (6) The use of the nonionic surfactant Triton X-100 leads to a complete loss of lipids and ubiquinone of the bc1-complex. (7) The complex contains no succinate dehydrogenase as indicated by the absence of the 69 kdalton subunit in the dodecyl sulfate gel electrophoresis. In addition, it lacks an ubiquinone cytochrome c reductase activity and other electron transferring activities. This may be inferred from an inhibition by antimycin and depletion of ubiquinone and phospholipids. The highly purified and relative stable complex can be prepared giving 50% yield and may be suitable for protein chemistry studies.


EPR signals of NADH: Q oxidoreductase. Shape and intensity. (1) The EPR spectrum of Center 1 of NADH dehydrogenase in isolated Complex I or submitochondrial particles from beef heart consists of two overlapping nearly axial signals of the same intensity. They are defined as Center 1a (gll = 0.021, gl = 1.938) and Center 1b (gll = 2.021, gl = 1.928). (2) The line shape of the EPR spectrum of the Center 3+4 can be interpreted as an overlap of two rhombic signals of the same intensity. We define Center 3 by the g-values: gz=2.103, gy = 1.93-1.94, gx=1.884, and Center 4 by the values gz=2.04, gy=1.92-1.93, gx=1.863. (3) Direct quantitation of the individuals signals as well as computer stimulation suggests that the amount of the Centers 1a and 1b is only 25% of that of the other individuals centers and FMN. As EPR spectra of beef-heart submitochondrial particles at 10-20 K are nearly identical to those of Complex I, the same relative concentrations of the Fe-S centers are also present in the particles. (4) The signals either observed by us in EPR spectra of Complex I and submitochondrial particles at 4.2 K and high microwave powers can now be explained without assuming more than 5 paramagnetic centers in NADH dehydrogenase.


Outer membrane of Salmonella typhimurium. Electron spin resonance studies. The supramolecular structure of the outer membrane of Salmonella typhimurium that produces an Rc-type lipopolysaccharide was studied by adding spin-labeled fatty acid probes to membranes as well as model bilayers. Lipopolysaccharide of this organism apparently formed a bilayer structure in 0.2 M NaCl/0.01 M MgCl2, and the electron spin resonance spectra suggested that the motion of the segments of hydrocarbon chains near the carboxyl end was quite restricted even at high temperature; this is presumably due to the anchoring of more than a dozen fatty acid residues to a single backbone structure. In the presence of Mg2+, we could produce lipoplysaccharide-phospholipid mixed bilayers contining up to 50% (by weight) lipoplysaccharide. Their spectra showed no sign of major heterogeneity, and the maximum hyperfine splitting values were considerably larger than in phospholipid-only liposomes; these results suggest that the two components are finely interspersed and that the mobility of phospholipid hydrocarbons is severely restricted by the hydrocarbon chains of lipopolysaccharide. In spite of the presence of lipoplysaccharide in an amount equal to or exceeding that of phospholipids, the outer membrane produced spectra remarkably similar to those of the inner membrane, which does not contain lipoplysaccharide, and there was little sign of immobilization by lipopolysaccharides. Signals corresponding to the pure lipoplysaccharide phase were not detected, either. These results suggest that the phospholipids and lipopolysaccharides are segregated into separate domains in the outer membrane, and the fatty acid probes enter almost exclusively into the phospholipid domains. This conclusion was fully corroborated by determining, through the exchange broadening of line width, the total area of the domains that accommodated the spin label probes.


Prostaglandin receptor-adenylate cyclase system in plasma membranes of rat liver and ascites hepatomas, and the effect of GTP upon it. 1. Adenylate cyclase in plasma membranes from rat liver was stimulated  by prostaglandin E1, and to a lesser extent by prostaglandin E2. Prostaglandin F1alpha and A1 did not stimulate the cyclase. The prostaglandin E1-mediated activation was found to require GTP when the substrate ATP concentration was reduced from 3 mM to 0.3 mM in the  reaction mixture. Adenylate cyclase of the plasma membranes from rat ascites hepatomas AH-130 and AH-7974 was not stimulated by prostaglandin E1 in the presence or the absence of GTP, although the basal activity of adenylate cyclase as well as its stimulation by GTP alone were similar to normal liver plasma membranes. 2. Liver plasma membranes were found to have two specific binders for [3H] prostaglandin E1 with dissociation constants of 17.6-10(-9) M and 13.6-10(8) M (37 degrees C) and one specific binder for [3H]prostaglandin F2alpha with a dissociation constant of 2.31-10(8) M (37 degrees C). The specific binders for prostaglandin E1 could not be detected in the hepatoma plasma membranes. 3. Binding of [3H] prostaglandin E1 to the liver plasma membranes was exchange by, GTP dGPT, GDP, ATP and GMP-P(N)P, but not by GMP, CGMP, DTTP, UTP or CTP. The increase in the binding of [3H] prostaglandin E1 was found to be due to the increased affinity of the specific binders to prostaglandin F2alpha was not affected by GTP. 4. GTP alone was found to increase V of adenylate cyclase of liver plasma membranes, while GTP plus prostaglandin E1 was found to decrease Km of adenylate cyclase in addition to the increase of V to a further extent.


Magnetic resonance study of the distribution of 2,2,6,6-tetramethylpiperidine-N-oxyl in phosphatidylcholine bilayers. With the aid of paramagentic praseodymium ions the resonances at 60 MHz of the inward and outward facing choline methyl protons of sonicated egg yolk phosphatidylcholine vesicles were resolved. The subsequent addition of 2,2,6,6,-tetramethylpiperidine-N-oxyl (TEMPO) to the vesicle suspension broadened the inner and outer resonances equally. TEMPO easily penetrates the egg yolk phosphatidylcholine vesicles and has free access to the entire lipid volume above the gel to liquid crystalline transition temperature. The electron spin resonance (ESR) spectrum of TEMPO in the egg yolk phosphatidylcholine suspension exhibits features indicating that TEMPO dissolves principally in the hydrocarbon portion of the egg yolk phosphatidylcholine bilayer. The egg yolk phosphatidylcholine methylene chain proton resonances are also broadened by TEMPO notably to a greater extent than the choline methyl resonances. These data indicate that TEMPO should be more sensitive to the melting behavior of the fatty acyl chains of phospholipid suspensions than to the polar head groups.


Cooperativity of the phase transition in single- and multibilayer lipid vesicles. The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.


Studies on bovine adrenal estrogen sulfotransferase. III. Facile synthesis of 3'-phospho- and 2'-phosphoadenosine 5'-phosphosulfate. A practical synthesis of 3'-phosphoadenosine 5'-phosphosulfate (IV) in yields of 68-72% from adenosine 2',3'-cyclic phosphate 5'-phosphate (II) is described. Reaction of II with triethylamine-N-sulfonic acid affords adenosine 2',3'-cyclic phosphate 5'-phosphosulfate (III) which, on treatment with ribonuclease-T2, provides IV. Spleen phosphodiesterase, on the other hand, converts III to 2'-phosphoadenosine 5'-phosphosulfate (V). The biological activity of IV, measured by sulfate transfer to [6,7-3H2]estrone as mediated by bovine adrenal estrone sulfotransferase (3'-phosphoadenylyl-sulfate:estrone 3-sulfotransferase, EC 2.8.2.4), is identical with that obtained with a sample of IV prepared by an established biochemical procedure. By contrast, V exhibits approximately one-third the activity of the natural isomer.


Cyclic nucleotide phosphodiesterases in the cricket, Acheta domesticus. Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second peak specific for cyclic AMP. There were no appreciable changes in the specific activity or kinetic properties of accessory gland cyclic GMP phosphodiesterase during a developmental period over which cyclic GMP levels rise more than 500-fold. Thus, the accumulation of cyclic GMP in the accessory gland is probably not associated with concomitant developmental modulation of phosphodiesterase activity.


EPR determination of the Co(II)-free radical magnetic geometry of the "doublet" species arising in a coenzyme B-12-enzyme reaction. The physical significance of the observed structure of the EPR signal, commonly known as the "doublet" spectrum, is that it contains information not only about the exchange coupling but also about the geometry of the magnetic dipole-dipole spin-spin coupling. We can show this because we have developed a general method of analysis applicable to this type of system and because we demand a quantitative fit of theory to experiment at two microwave frequencies. We have chosen the "doublet" free radical signal, which arises in the ribonucleotide reductase-5'-deoxyadenosylcobalamin system (from Lactobacillus leichmannii, see Hamilton et al., Biochemistry 11, 4696--4705 (1972)), for study, for the particular reason that the 35 GHz "doublet" spectrum has three components (in this case) rather than two, which provides an important test of the recently proposed model of isotropic exchange coupling by Schepler et al. ((1975) Biochim. Biophys. Acta 397, 510--518). We find that a quantitative fit to the EPR "doublet" lineshape can be obtained with a model of isotropic exchange, and a "point" magnetic dipole-dipole interaction acting over a distance of 9.9 A with the radical located approx. 34 degrees off the principal gzz axis and less than 1 degree off the principal gxx axis of the Co(II) in the corrin ring. Quantitative fits of the doublet portion of the observed lineshape at both 9 and 35 GHz were achieved with this model, assuming an axially symmetric free radical signal and a Gaussian spin-packet lineshape with isotropic linewidth.


Chloroquine-induced interference with degradation of serum lipoproteins in rat liver, studied in vivo and in vitro. The effect of chloroquine, an inhibitor of certain lysosomal enzymes including cathepsin B (EC 3.4.22.1), on the degradation of serum lipoproteins in rat liver was studied in vivo and in liver homogenates. Chloroquine had no effect on the clearance from the circulation of 125I-labeled rat or human very low density lipoproteins or human low density lipoproteins. Pretreatment with chloroquine for 3 h, resulted in a 2-2.5 fold increase in 125i-labeled very low density lipoprotein recovered in the liver 45 min after injection of the homologous and heterologous lipoproteins. This effect was evident on both the 125I-labeled protein and 125I-labeled lipid moiety. 30 min after the injection of [3H]-cholesterol linoleate-labeled very low density lipoproteins, 70% of the injected label was recovered in the liver, both in control and chloroquine-treated rats. Since the perl and 20% in the experimental group, it was concluded that chloroquine interferes with the hydrolysis of [3H]cholesterol linoleate. Following injection of 125I-labeled human low density lipoproteins only 4% of the injected lipoprotein was recovered in the liver of control rats and not more than 10% after chloroquine treatment, when about 50% had been cleared from the circulation. Hence, while very low density lipoprotein protein and cholesterol ester are catabolized in the liver, the catabolism of low density lipoproteins occurs mainly in extra-hepatic tissues. Using post-nuclear liver suprnatant, optimal degradation of various serum lipoproteins was found at pH 4.4, and chloroquine inhibited their degradation. Degradation of very low density and low density lipoproteins was completely inhibited at 0.05 M chloroquine, while less pronounced inhibition was seen with high density lipoproteins, apolipoproteins and apolipoprotein AI. These results indicate that liver acid hydrolases in vivo participate in the degradation of serum lipoproteins. Cathepsin B is apparently responsible for the degradation of aplipoprotein B, while other cathepsins might also be active in the degradation of this and the other apolipoproteins.


Partial purification and characterization of NAD-dependent 7alpha-hydroxysteroid dehydrogenase from Bacteroides thetaiotaomicron. A NAD-dependent 7alpha-hydroxysteroid dehydrogenase was purified 18-fold over the activity in crude cell extracts prepared from Bacteroides thetaiotaomicron NCTC 10852 using Bio-Gel A 1.5-M column chromatography. A molecular weight of 320 000 was estimated for the partially purified intact enzyme. Substrate saturation kinetics were performed using the 18-fold purified enzyme and the lowest Km values were obtained for 3alpha,7alpha-dihydroxy bile acid and bile salt substrates including chenodeoxycholic acid (Km 0.048 mM), glycochenodeoxycholic acid (Km 0.083 mM) and taurochenodeoxycholic acid (Km 0.059 mM). In contrast, 3alpha,7alpha,12alpha-trihydroxy bile acid and bile salts had higher Km values, i.e. cholic acid (Km 0.22 mM), glycoholic acid Km 0.32 mM) and taurocholic acid Km 0.26 mM). NAD had a Km value of 0.20 mM. The possible physiological significance of 7alpha-hydroxy bile acid oxidation to intestinal bacteroides strains was accessed by determining the rate of conversion of [14C]-cholic acid to 7-ketodeoxy[14C]cholic acid by whole cell suspensions under different incubation conditions. The rate of biotransformation of bile acid to keto-bile acid incubated anaerobically under N2 gas increased markedly when potential electron acceptors such as fumarate (10 mM) or menadione (4 mM) was added exogenously. These results suggest that bile acid oxidation reactions may be linked to energy-generating systems in this bacterium.


Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells. The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.


Radioimmunoassay of arginine-rich apolipoprotein of rat serum. A double-antibody radioimmunoassay was developed for quantification of rat arginine-rich apolipoprotein in sodium decyl sulfate. Arginine-rich protein, labeled with 125I by the chloramine-T method, had the same chromatographic characteristics on Sephadex G-200 as unlabeled arginine-rich protein and up to 70% of 125I-labeled arginine-rich protein was precipitated by antisera to arginine-rich protein in rabbits. The assay is sensitive at the level of 1-10 ng and has intraassay and interassay coefficients of variation of 5.4 and 6.8%, respectively. The specificity of the assay was established by competitive displacement of 125I-labeled arginine-rich protein from its antiserum by arginine-rich protein and lipoproteins containing this protein, but not by rat albumin or other purified apolipoproteins. Immunoreactivity of rat serum and lipoproteins was complete as demonstrated by comparison with their delipidated form. The accuracy of the immunoassay was further substantiated by comparison with the amount of arginine-rich protein in chromatographic fractions of total apoprotein of very low and high density lipoproteins, and by recovery experiments in ultracentrifugally separated fractions of serum. In contrast to an immunoassay reported previously for rat apo A-I, sodium decyl sulfate was not required for complete immunoreactivity of serum and lipoproteins. The inclusion of sodium decyl sulfate (9 mM final concentration) was necessary, however, for stability of labeled and unlabeled preparations of arginine-rich protein. Content (weight %, means values +/- S.D.), of immunoassayable arginine-rich protein in isolated lipoproteins was 15 +/- 1.5% in very density lipoproteins; 6.8% in low density lipoproteins (1.02 less than d less than 1.04 g/m); 7.1 +/- 0.3% in high density lipoproteins; and 4.8 +/- 0.5% in lymph chylomicrons. Concentration in whole serum was 18.1 +/- 1.4 and 20.4 +/- 2.3 mg/dl for male and female rats, respectively. Only about 55% of arginine-rich protein was recovered in the major lipoprotein classes and about 40% was in "lipoprotein-free" serum (d greater than 1.25 g/ml). Among the lipoproteins, the high density lipoprotein fraction contained twice the amount of arginine-rich protein recovered in very low or low density lipoproteins (26.6 vs. 13.5 and 13.4%, respectively). The significance of the large amount of arginine-rich protein in the 1.25 g/ml infranatant fraction is not apparent. Although repetitive centrifugation did not alter the amount recovered in this fraction, the possibility of an artifact induced by centrifugation and high salt concentration cannot be excluded.


Solvent denaturation of globular proteins: unfolding by the monoalkyl- and dialkyl-substituted formamides and ureas. The effects of the monoalkyl and dialkyl-substituted formamide series of denaturants on the native conformation of sperm whale myoglobin, horse heart cytochrome c, and Glycera dibranciata (single chain) hemoglobin have been investigated by spectral measurements in the Soret region (409 and 422 nm) and optical rotation measurements (265nm). The effectiveness of these two classes of protein denaturants is similar to the other straight-chain compounds of the urea, amide, and alcohol classes, examined in previous investigations from our laboratory. Their denaturing effectiveness is found to increase with increasing chain length or hydrocarbon content of the substituent alkyl groups. Application of the Peller and Flory equation to the denaturation data of the formamides shows that both the polar and the nonpolar group contributions to the protein-denaturant interactions have to be taken into account in order to correctly predict the observed denaturation midpoints. Additivity of the hydrophobic, KHø, and the polar, Kp, group contributions to the binding constants, KB = nKHø + Kp, with n = 1 or 2 for the mono- of the di-alkyl substituted denaturants gave best account of the experimental data. The KHø values used were based on free energy transfer data of various alkyl groups or the Scheraga-Nemethy theory of hydrophobic bonding. The assumption of group contributions of the denaturant to KB were also applied to the denaturation data of the unsubstituted amides and some examples of the monoalkyl and symmetrically substituted dialkyl ureas, taken from the literature.


Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives. The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.


An EPR study of the lineshape of copper in cytochrome c oxidase. The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220 degreesK, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as gx = 1.99, gy = 2.03, gz = 2.185, / Ax(Cu) / = 0.0020 cm-1, / Ay(Cu) / = 0.0025 cm-1, / Az(Cu) / = 0.0030 cm-1. The principal values of the g tensor and the small value of /Az(Cu) / are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu2+ with an additional rhombic distrotion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values deltaHx = 45G, deltaHy = 65 G, deltaHz = 85 G. A weak dipolar interaction with a low-spin ferric species stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time has been observed near g = 14 and g = 3 at 5 degrees K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the "EPR-undetectable" iron and copper is raised.


The application of ESR to chromatin. Spin-labelling of the histone H3 cysteine residues in situ. The possibility has been investigated of selectively spin-labelling the cysteine residues of histone H3 in chromatin and probing by ESR conformational changes affecting the labelled area as the molecular environment is altered. About 90% of bound labels are attached to the thiol groups and are strongly immobilized in deep crevices. The remaining labels are bound to amino groups mainly on histone H1, giving rise to a more mobile component in the chromatin spectrum. No conformational changes involving the labelled cysteins could be detected as the histones were dissociated stepwise from the complex by NaCl, but treatment with urea led to a cooperative increase in mobility, indicating that the hydrophobic region around the cysteine residues is folded in a compact tertiary structure to which histone H4 may be bound in the native complex, but which is not affected by dissociation of the H3-H4 unit from the DNA. In addition, chymotryptic disruption of the chromatin has been followed and an estimate made from the rotational correlation times of the size and origin of the digestion fragment carrying spin-labelled cysteine 110.


High resolution EPR studies of the fine structure of heme proteins. Third harmonic detection approach. When third harmonic detection is applied in EPR studies of the superhyperfine structure of nitrosyl derivatives of a number of human hemoglobin variants, significant resoltuion enhancement is obtained. This has allowed a detailed analysis of the number of superhyperfine lines, their g-values and their splittings, and has led to a more complete understanding of the interaction between the axial ligands of the heme iron. Sudies of the effect of the modulation amplitude on EPR line-shapes revealed that the amplitude required to resolve fine structure in the third harmonic mode is 3-10 times larger than that used to record an undistorted first derivative spectrum. The application of this approach for other systems is discussed, and practical guidelines for its use are given.


Crossing of low-lying electronic levels of high-spin ferrous ion in deoxyhemoglobin and deoxymyoglobin. The electronic structure of high-spin S = 2 ferrous ion in deoxy forms of hemoglobin and myoglobin is considered in terms of spin Hamiltonian formalism. Spin Hamiltonian parameters of the second order B0(2)(D), B2(2)(E) and, for the first time in the available literature, of fourth order B0(4), B2(4) and B4(4), are calculated for the rhombic symmetry case of Fe2+. The Hamiltonian matrix is diagonalized for several sets of Bq(k) parameters compatible with other experimental data. The low-lying Fe2+ levels exhibit crossings in a high magnetic field, applied along the z-axis perpendicular to the heme plane. The cross-over values of the magnetic field are determined to be Hc1 = 46 kOe and Hc2 = 168 kOe for D = 5.2, E = 0.6 cm-1 (close to the magnetic data of Nakano, N., Otsuka, J. and Tasaki, A. (1972) Biochim. Biophys. Acta 278, 355-371) and with B0(4) = 0.037, B2(4) = 0.005, B4(4) = 0.013 cm-1 and gz = 2.028. Experimental techniques for measurement of the crossing effects are discussed.


Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas. Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdII. The g = 1.94 signal seen in dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdII. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdII. It is concluded that most of the 4Fe-4S centres in FdI change between states C- and C2-; FdI' contain both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdII even in the diamagnetic C2- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdII and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g = 1.94 signal in the reduced states.


Physical, chemical and immunochemical characterization of a lipoprotein lipase activator protein from pig plasma very low density lipoproteins. Very low density lipoproteins ere isolated from plasma of swine by ultracentrifugal flotation. After delipidation, the lipid-free proteins were separated by chromatography on Sephadex G-150 AND DEAE-cellulose. A major apoprotein was isolated and shown to activate cows' milk lipoprotein lipase. Since human very low density lipoproteins also contain an activator protein, designated, apoC-II, we have called the pig protein, pig apoC-II. Pig apoC-II had a molecular weight of approximately 10 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid composistion showed the absence of histidine, cysteine and tryptophan; there was no evidence for carbohydrate. Treatment of pig apoC-II with carboxypeptidase indicated COOH-terminal serine. Rabbit antisera prepared to the pig protein gave single precipitin lines of complete identity to very low density lipoproteins, apoC-11. Using anti-pig apoC-II, a radioimmunoassay was developed which provides a convenient and reproducible method for measuring 5-1000 ng of apoprotein.


Chemical analysis of the hyphal wall of Schizophyllum commune. 1. Purified hyphal wall fragments of Schizophyllum commune are analysed and shown to consist of glucose (67.6%), mannose (3.4%), xylose (0.2%), (N-acetyl)glucosamine (12.5%), amino acids (6.4%) and some lipid material (3.0%). 2. The previously proposed structures of two glucans located at the hyphal wall surface (Wessels et al. (1972) Biochim. Biophys. Acta 273, 346-358) were essentially confirmed using methylation analysis. The mucilaginous glucan consists of 1,3-linked beta-glucan chains with branches of single glucose units attached by beta-1,6 linkages on every third unit, on average, along the chain. The alkali soluble S-glucan is an exclusively 1,3-linked alpha-glucan. 3. The alkali-insoluble R-glucan, occurring in close association with chitin, in the inner wall layer, has been characterised by methylation analysis, X-ray diffraction, enzymatic hydrolysis with purified exo-beta-1,3-glucanase and Smith degradation. It appears to be a highly branched beta-1,3,beta-1,6-glucan and a model of this glucan is proposed. Certain parts of this highly insoluble R-glucan bear a close structural similarity to the mucilaginous glucan present at the outer wall surface and in the medium.


Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release. 1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin  and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.


Insulin-sensitive adenosine 3',5'-cyclic monophosphate phosphodiesterase of hepatocyte plasma membrane. Adenosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.17) has been investigated in rat liver as to its insulin sensitivity. Hormone action has been assayed in vitro on a liver homogenate purified by DEAE-cellulose column chromatography, on isolated hepatocytes, on isolated plasma membranes. The DEAE-cellulose chromatography purified homogenate showed no sensitivity to insulin, whereas isolated hepatocytes incubated in presence of insulin showed increased phosphodiesterase activity in a plasma membrane-containing fraction. The plasma membrane-bound enzyme, which shows both high and low affinity components, was significantly stimulated after hormonal treatment; this effect being dependent on a V increase of the low Km form.


Effect of diabetes and insulin on rat renal glomerular protocollagen hydroxylase activities. The effect of diabetes and insulin on the activities of both prolyl hydroxylase (trivial name; proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) and lysyl hydroxylase (trivial name; lysine,2-oxoglutarate dioxygenase, EC 1.14.11.4) in isolated rat renal glomeruli was determined. Three groups of experimental animals were used: age-matched controls, streptozotocin-diabetic, and insulin-treated streptozotocin-diabetic. Using 14C-labeled lysine or proline hydroxylase substrate prepared from chick embryo tibiae, glomerular 17 000 X g supernatant enzyme was incubated in a complete hydroxylating system for 60 and 120 min Lysyl hydroxylase activity was significantly increased in diabetic preparations, but prolyl hydroxylase activity did not differ from control. Administration of insulin to streptozotocin-injected animals completely restored glomerular lysyl hydroxylase to normal levels. The results suggest that the specific elevation of lysyl hydroxylase relates to the biochemical changes contributory to diabetic nephropathy, and that insulin may reverse this process.


Studies on the mechanisms of epinephrine stimulation of lypolysis. The time course for epinephrine stimulation of lypolysis, cyclic AMP accumulation and activation of protein kinase was studied in adipose tissue from hypophysectomized rats. Triglyceride breakdown, as assessed by glycerol release, increased rapidly in response to epinephrine, maintained a constant rate as long as the hormone was present, and decreased rapidly to basal values when the hormone was removed. Cyclic AMP accumulation was transient peaking within 3 min of exposure to epinephrine and then declining to levels indistinguishable from basal by 9 min. Protein kinase activity in extracts also peaked at 3 min and thereafter declined to a level approximately 25% greater than resting activity. Peak levels of cyclic AMP, steady state levels of protein kinase activity and the rate of glycerol production were all related in a dose dependent manner to the concentration of epinephrine. These observations suggest that the spike in cyclic AMP levels may be necessary to trigger the activation of lipolysis, but was not sufficient to sustain an accelerated rate of triglyceride breakdown. Continued activation of protein kinase, however, may be essential to sustained lipolysis.


Preparation and characterization of specifically tritiated adrenocorticotropin. The catalytic dehalogenation of iodinated derivatives of corticotropin in the presence of tritium was investigated. In 0.1 M acetic acid, complete and rapid removal of iodine was achieved in the presence of freshly prepared palladium or palladium oxide as catalyst, but the specific radioactivity of the product was only 10-20% of the theoretically attainable value. Synthetic human corticotropin containing a 3,5-diiodo tyrosine in position 23 in place of tyrosine was successfully dehalogenated in solvent mixture 0.1 M acetic acid: hexamethylphosphoramide: dimethylformamide (1 : 10 : 90, v/v) in the presence of palladium oxide and calcium carbonate. The product was obtained in 30% yield after purification by carboxymethyl cellulose chromatography. The tritiated hormone had a specific radioactivity of 46 Ci/mmol (80% of the theoretical value) and was as potent as synthetic human corticotropin in stimulating steroidogenesis and lipolysis.


On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells. The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.


Evidence for nuclear and DNA binding forms of the receptor-glucocorticoid complex from hepatoma tissue culture cells. Binding to DNA associated with cellulose has been used to investigate the receptor-glucocorticoid complex isolated from a line of rat hepatoma tissue culture cells. The amount of activated complex that bound to DNA was approximately half that which bound to nuclei. Additional results suggest the existence of two forms of the activated glucocorticoid receptor-steroid complex in about equal amounts: one form binds only to nuclei and the other binds to DNA and nuclei. The two forms also differ in their stability, with the DNA/nuclei binging form being relatively labile. The binding of either form to the appropriate acceptor is reduced by cytosol inhibitors by the same mechanism.


Glucocorticoid receptor-steroid complex binding to DNA. Competition between DNA and DNA-cellulose. The binding of the glucocorticoid receptor-steroid complex from a line of rat hepatoma tissue culture (HTC) cells to DNA has been examined. An equilibrium competition assay involving a constant, low total amount of double-stranded DNA was developed to compare the complex binding ability of DNA free in solution and bound to cellulose. This binding ability is lowered by a factor of five when DNA is associated with cellulose. Similar studies with HTC cell, calf-thymus, and Escherichia coli DNA revealed no difference in the relative number or affinity of binding sites for receptor-steroid complex in each DNA. The synthetic DNA molecules poly[d(A-T)-d(A-T)] and poly[d(G-C)-d(G-C)] bound complexes equally well but less than the three "natural" DNA molecules. This appears to be due to differences in acceptor site affinity and suggests that nucleotide complexity and/or sequence influences the affinity of HTC cell receptor-glucocorticoid complexes for DNA.


Immunoassay for honey bee cytochrome c in single animals with cytochrome c-coated bacteriophages: a sensitive tool for the study of caste formation in the honey bee, Apis mellifera. The development of a sensitive viroimmunoassay for honey bee cytochrome c and its usage for early detection of caste differentiation is described. Pure honey bee cytochrome c was isolated from workers and used to produce antibodies in rabbits. Bacteriophage T4 was chemically modified by covalent attachment of honey bee cytochrome c using tolylene-2,4-diisocyanate as a cross-linking agent. The immunospecific inactivation of this bacteriophage-cytochrome c conjugate by anti-cytochrome c antibodies can be inhibited by free cytochrome c. In quantitative determinations, 50% inhibition is reproducibly achieved at a concentration of 6 ng/ml (5 pmol/ml) and as little as 0.3 ng/ml (0.25 pmol/ml) could be detected by this system. Cytochrome c concentrations were measured in individual animals and substantial differences corresponding larval stages of worker and queen bees are reported.


Genetic evidence for the common identity of glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamyl phosphate-glucose phosphotransferase and inorganic pyrophosphatase. We demonstrate that glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamyl phosphate-glucose phosphotransferase and inorganic pyrophosphatase activities are deficient in livers of patients with type I glycogen storage disease. This provides strong genetic evidence that these enzymatic activities reside in a single protein or share a common polypeptide chain.


Effect of metabolites and phosphorylase on the D to I conversion of glycogen synthase from human polymorphonuclear leukocytes. The D to I conversion of glycogen synthase from human polymorphonuclear leukocytes was examined both in a gel-filtered homogenate and in a preparation of glycogen particles with adhering enzymes, purified by chromatography on concanavalin A bound to Sepharose. It was found that glucose 6-phosphate as well as mannose 6-phosphate, glucosamine 6-phosphate, and 2-deoxy-glucose 6-phosphate activated the reaction, whereas the corresponding sugars were without effect. Mn2+ and Ca2+ increased the conversion rate by 51% and 27%, respectively, whereas Mg2+ and inorganic phosphate were without effect. Sodium fluoride inhibited the reaction completely. Glycogen inhibited the reaction in physiological concentrations and 0.5 mM glucose 6-phosphate was able to overcome this inhibition. MgATP greatly augmented the inhibition caused by glycogen in the glycogen particle preparation. This combined effect could be overcome by glucose 6-phosphate in concentrations from 0.1 to 1 mM. Phosphorylase alpha purified from human polymorphonuclear leukocytes inhibited the D to I conversion in a glycogen particle preparation. The inhibition was counteracted by glucose 6-phosphate and to a lesser degree by AMP. Phosphorylase beta was also inhibitory, but only at higher concentrations than phosphorylase alpha. No phosphorylase phosphatase activity was found in the glycogen particle preparation, which may indicate that chromatography on concanavalin A-Sepharose separates this enzyme from the synthase phosphatase or partially destroys the activity of a hypothetical common protein phosphatase.


On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase. We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an alpha-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase b kinase (EC 2.7.1.38). (b) The absence of Ca2+ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca2+ was present in the incubation medium. (d) Glucagon, cyclic AMP and three cyclic AMP-dependent hormones caused an enhanced uptake of 45Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate 45Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced 45Ca uptake and the phosphorylase activation. We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca2+ is most probably phosphorylase b kinase.