Biomedical subjects
Publications and source records attributed to M Vaughan.
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Procedures for the determination of free and total cholesterol in lipid extracts or sonicates of 10(4) cultured human skin fibroblasts are described. The method for free cholesterol employs cholesterol oxidase to generate H2O2 and peroxidase to catalyze the reaction of H2O2 with p-hydroxyphenylacetic acid to yield a stable fluorescent product. Cholesterol ester hydrolase is included for determination of total cholesterol. When samples of sonified cell suspensions are used directly, the extraction of lipids is avoided, permitting one person to carry out analyses of 30 or more subcultures in one day.
Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.
Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.
Choleragen catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide; nicotinamide production was dramatically increased by L-arginine methyl ester and to a lesser extent by D- or L-arginine, but not by other basic amino acids. Guanidine was also effective. Nicotinamide formation in the presence of L-arginine methyl ester was greatest under conditions previously shown to accelerate the hydrolysis of NAD by choleragen (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4424-4427). After incubation of [adenine-U14C]NAD and L[3H]arginine with coleragen, a product was isolated by thin layer chromatography that contained adenine and arginine in a 1:1 ratio and has been tentatively identified as ADP-ribose-L-arginine. Parallel experiments with [carbonyl-14C]NAD have demonstrated that formation of the ADP-ribosyl-L-arginine derivative was associated with the production of [carbonyl-14C]nicotinamide. As guanidine itself was active and D- and L-arginine was equally effective in promoting nicotinamide production, whereas citrulline, which possesses a ureido rather than a guanidino function, was inactive, it seems probable that the guanidino group rather than the alpha-amino moiety participated in the linkage to ADP-ribose. Based on the assumption that the ADP-ribosylation of L-arginine by choleragen is a model for the NAD-dependent activation of adenylate cyclase by choleragen, it is proposed that the active A protomer of choleragen catalyzes the ADP-ribosylation of an arginine, or related amino acid residue in a protein, which is the cyclase itself or is critical to its activation by choleragen.
DEAE-Bio-Gel chromatography of 100,000 X g supernatant from cultured HTC hepatoma cells separated cyclic nucleotide phosphodiesterase into three forms, numbered E I, E II, and E III in order of elution from the column, E I had a low Km for cyclic guanosine 3':5'-monophosphate (cGMP) and a high Km for cyclic adenosine 3':5'-monophosphate (cAMP), E II exhibited anomalous kinetics. At low substrate concentrations (0.5 muM) cGMP was hydrolyzed more rapidly than cAMP and hydrolysis of 0.5 muM cAMP was stimulated by 1 muM cGMP. E III had a low Km for cAMP. Incubation of cells with 1 muM dexamethasone for 72 h decreased the activity of E I and E II. In cells incubated with N6,O2'-dibutyryl cAMP plus 3-isobutyl-1-methylxanthine for 14 h the activity of E III was increased approximately 100%. Similar activities of calcium-dependent, heat stable phosphodiesterase activator were recovered from supernatants from all cells. These studies have established the presence, in a homogeneous population of hepatoma cells, of at least three forms of cyclic nucleotide phosphodiesterase, the activities of which can be independently regulated.
Choleragen and its A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. NADase activity was inhibited by gangliosides GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM3 (N-acetylneuraminyl-galactosylglucosylceramide), and GD1a (N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-E1N-acetylneuraminyl]-galactosylglucosylceramide). These gangliosides also increased the intensity of the tryptophanyl fluorescence of the isolated A protomer (lambda max = 328 nm). GM1 but not GM2, GM3, and GD1a caused a "blue shift" in the fluorescence spectrum of the B protomer. These results are consistent with other evidence that the specificity of GM1 as the choleragen receptor resides in its carbohydrate moiety. The NADase activity of choleragen was similar to that of diphtheria toxin previously described [J. Kandel, R. J. Collier & D. W. Chung (1974) J. Biol. Chem. 249, 2088-2097]. As with diphtheria toxin, analogues of NAD were inhibitory, adenine being the most effective. Significant inhibition was also noted with adenosine, AMP, ADP-ribose, nicotinamide, nicotinamide mononucleotide, and NADP. NADP was hydrolyzed only slowly by choleragen. In the NADase reaction catalyzed by diphtheria toxin, water serves as an acceptor for the ADP-ribose moiety of NAD in lieu of the natural acceptor molecule, which is elongation factor II (Kandel et al., 1974). It seems probable that the natural protein acceptor for ADP-ribose in the reaction catalyzed by choleragen is adenylate cyclase or a protein component of a cyclase complex that regulates enzymatic activity.
The requirements for choleragen activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] were investigated by using an enzyme preparation solubilized with Triton X-100 from an extensively washed brain particulate fraction and partially purified with DEAE-cellulose. Unlike the particulate enzyme, this preparation was not activated after incubation with choleragen plus dithiothreitol, ATP, and NAD. Addition of the purified protein activator of cyclic nucleotide phosphodiesterase and calcium to the partially purified enzyme increased basal activity somewhat, but choleragen activation was minimal. When cyclase was incubated with GTP plus the protein activator (and calcium), choleragen markedly increased the activity 3- to 6-fold. When GppNHp and protein activator were incubated with the cyclase prior to assay, activity was elevated but no effect of choleragen was observed. GTP and GppNHp had relatively small effects on cyclase activity in the absence of protein activator or if they were added directly to the assay. Boiled brain supernatant was consistently more effective than protein activator (plus calcium) and GTP, suggesting that other factors are required for maximal cyclase activity after choleragen treatment. It appears that the cyclase system is dissociable into several components, all of which may be necessary for optimal regulation of activity. It is probable that one of these is the heat-stable calcium-dependent protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase that we have found is required along with GTP for demonstration of choleragen activation of partially purified brain adenylate cyclase.
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Thirteen patients diagnosed as having affective disorders, and who were taking lithium, were compared with drug-free controls on short-term and long-term memory tasks. There was some indication that patients on lithium may show an impairment of short-term memory at fifteen-second delay intervals, and possibly enhanced long-term recall of difficult material. Further comparison with results obtained from six patients on tricyclic antidepressants seemed to reduce the possibility that the lithium group's scores were a function of their psychiatric status. As the group sizes were small, all the findings need to be replicated.
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Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.
Incubation (30 degrees) of fat cell particulate fractions with fluoride before assay in the effective absence of fluoride results in activation of adenylate cyclase. Whereas the effect of fluoride (1.3 to 7 mM) when added to the assay was maximal in less than 2 min, 10 or 15 min of incubation before assay was usually required to produce maximal activation with any given concentration of fluoride. Under both conditions 3 to 5 mM fluoride produced maximal activation. After incubation with fluoride for 5 to 20 min cyclase activity was constant for at least 15 min of assay without fluoride; maximal activity was greater than that produced by fluoride added to the assay system and the concentration required to produce significant activation was lower. Fluoride activation in the assay or during prior incubation could be prevented by pyrophosphate. When added during the early minutes of assays with fluoride, 1.5 mM pyrophosphate, which had little effect on the activity of enzyme previously incubated with fluoride, rapidly reduced activity to essentially basal levels; when added after 10 min its effect was equally rapid but much smaller. It appears that activation is initially reversible by removal of fluoride as it is by addition of pyrophosphate, but becomes largely irreversible by these means with continued exposure to fluoride. Fluoride in the assay system inhibited cyclase activated by isoproterenol or choleragen or by incubation with fluoride prior to assay; inhibition, dependent on fluoride concentration, was maximal at 5.3 mM. Since maximal activity produced by incubation with fluoride before assay exceeded that of nonincubated preparations assayed with fluoride, and was reduced to the latter level when assayed in the presence of fluoride, we infer that inhibition is reversible at a time when fluoride activation is relatively irreversible. Pyrophosphate (1.5 mM), which prevented fluoride activation, did not reduce fluoride inhibition of isoproterenol-, fluoride-, or choleragen-activated cyclase. When 3 mM MnCl2 was present in the assay, inhibition by fluoride was not observed. In descriptive terms, MnCl2 appeared to cause rapid reversal of fluoride inhibition. Thus, fluoride inhibits, in an apparently similar manner, fat cell adenylate cyclase whether it is activated by isoproterenol, fluoride, or choleragen. Although fluoride activation and inhibition can apparently be dissociated or modified differentially, until the mechanism(s) of action of fluoride is elucidated it cannot be concluded that these are totally independent processes.
NCTC 2071 cells, transformed mouse fibroblasts, when grown in chemically defined medium, are deficient in gangliosides and do not respond to choleragen. The cells lack two biosynthetic enzymes, CMP-sialic acid:lactosylceramide sialyltransferase and UDP-galactose:GM2 (GalNAc-[AcNeu]-Gal-Glc-ceramide) galactosyltransferase, which are required for ganglioside synthesis. Following uptake of ganglioside GM1 (Gal-GalNAc-[AcNeu]-Gal-Glc-ceramide) from the medium, the cells respond to choleragen; however, they remain unresponsive following uptake of gangliosides GM2 (approximately 6 X 10(6) molecules/cell) and GM3 (AcNeu-Gal-Glc-ceramide) (approximately 2 X 10(6) molecules/cell). A response was observed when the cells had bound approximately 2 X 10(7) molecules of GM2/cell. After binding GD1a (AcNeu-Gal-GalNAc-[AcNeu]-Gal-Glc ceramide) (approximately 2 X 10(5) molecules/cell), cells exhibit some response to the toxin which can be attributed to enzymatic conversion of GD1a to GM1. A second line of NCTC 2071 cells which have 2.5 X 10(7) molecules of endogenous GM2/cell is slightly responsive to choleragen; adenosine 3':5'-monophosphate (cyclic AMP) levels rise 150%. However, when these cells have bound 4.4 X 10(4) molecules of GM1 per cell, cyclic AMP levels rise 7-fold following toxin treatment. GM1, which becomes functionally integrated into the cells, appears to be the natural receptor for choleragen and is 50 to 1000 times more effective than other gangliosides in eliciting a choleragen response.
125I-Labeled choleragen was bound to liposomes containing galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM1), but not in large amounts to ganglioside-free liposomes nor to those containing N-acetylneuraminylgalactosylglucosylceramide (GM3), N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM2), or N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD1a). Choleragen released trapped glucose only from GM1-liposomes. This choleragen-induced glucose release from GM1-liposomes was relatively rapid for the first few minutes, then continued more slowly. The amount of glucose released from liposomes in 30 min was dependent on both the GM1 content and choleragen concentration. Prior incubation of GM1-liposomes with anti-GM1 antiserum prevented the choleragen-dependent release of trapped glucose. After incubation of GM1-liposomes with choleragen, addition of anticholeragen antibodies and complement led to more extensive glucose release. Under these latter conditions a much smaller glucose release was observed also from liposomes containing GM1 or N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide in the absence of choleragen. These releases were attributed to naturally-occurring antiganglioside antibodies in the antiserum and complement. Ganglioside-free liposomes did not release glucose in response to anticholeragen and complement. It appears that choleragen in the absence of other proteins binds specifically to liposomes containing GM1 and can induce permeability changes.
Choleragen and the isolated A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. The protein with NADase activity (NAD nucleosidase; NAD glycohydrolase, EC 3-2-2-5) migrated on polyacrylamide gels with choleragen, and chromatographed on Bio-Gel P-60 columns with the A protomer. The NADase activity of choleragen and of the A protomer was increased markedly in acetate and phosphate buffers, and enhanced over 10-fold by dithiothreitol in high concentration. NAD hydrolysis was proportional to choleragen concentration; the Michaelis constant for NAD was about 4 mM with both choleragen and the A protomer. The demonstration that the A protomer of choleragen catalyzes an enzymatic reaction involving activation of the ribosyl-nicotinamide bond of NAD, a reaction analogols to those catalyzed by diphtheria toxin, supports the hypothesis that activation of adenylate cyclase by choleragen involves the ADP-ribosylation of an appropriate acceptor protein.
NCTC 2071 cells are unable to synthesize the monosialoganglioside GM1. When grown in chemically defined medium these cells contained no detectable GM1 and did not accumulate 3': 5'-cyclic AMP in response to choleragen. Incubation of the cells with [3H]GM1 permitted quantification of ganglioside uptake which was dependent on time and concentration of [3H]GM1 in the medium. Responsiveness to choleragen was demonstrated with binding of as few as 17,000 molecules of [3H]GM1 per cell; a maximal response was observed with 10(5) molecules per cell. With increasing cellular content of GM1, the rate of rise in intracellular cyclic AMP in response to choleragen was increased. With greater than 1 X 10(5) molecules of GM1 per cell, the delay between addition of choleragen and the cyclic AMP response was inversely proportional to choleragen concentration; less than 250 molecules of choleragen per cell caused a significant increase in cyclic AMP after 8 hr of incubation. Although the responsiveness of intact cells to choleragen was dependent on GM1, choleragen activation of adenylate cyclase in homogenates with 0.6 mM NAD was independent of added ganglioside. These observations are consistent with the view that exogenous ganglioside GM1 can be functionally integrated into the surface membrane of intact cells and serve as the choleragen receptor. Furthermore, although exogenous GM1 is required for choleragen responsiveness in intact cells, the ganglioside does not play an obligatory role in cell homogenates, where the surface receptor can presumably be bypassed.