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P R Clements

Publications and source records attributed to P R Clements.

33 records · Page 2Linked to original sources
PubMed1985

Human alpha-L-iduronidase. 2. Catalytic properties.

The kinetic parameters of human liver alpha-L-iduronidase were determined with three disaccharide substrates: alpha-L-iduronosyl(1----4)2,5-anhydro-D-[1-3H]mannitol 6-sulphate, alpha-L-iduronosyl(1----4)2,5-anhydro-D-[1-3H]mannitol and alpha-L-iduronosyl(1----3)2,5-anhydro-D-[1-3H]talitol 4-sulphate, derived from the natural substrates heparin and dermatan sulphate and one synthetic, fluorogenic substrate, 4-methylumbelliferyl alpha-L-iduronide. The enzyme activity with all four substrates was optimal at about pH 4.5. The Km values derived using the disaccharide substrates were elevated up to 10-fold with up to a 6.5-fold increase in ionic strength whereas that for the synthetic substrate was only increased by 1.7-fold. The V values for all substrates were unaffected. The inhibitory effect of NaCl, Na2SO4, NaH2PO4 or CuCl2 on enzyme activity was more pronounced with the disaccharide substrates than with the synthetic substrate. The moiety which is most important in binding is the idopyranosyl residue. While the aglycone residue adds to the net affinity for the enzyme, it is the substituent groups of both residues which appear to control catalysis. Specifically the carboxyl moiety of the alpha-L-iduronic acid residue is essential for catalysis while the presence of sulphate on the C4 or C6 position of the aglycone residue has a major influence on catalysis rather than binding. alpha-L-Idosyl(1----4)2,5-anhydro-D-[1-3H]mannitol 6-sulphate did not undergo catalysis and was a potent inhibitor of enzyme activity, whereas beta-glucuronosyl(1----4)2,5-anhydro-D-[1-3H]mannitol 6-sulphate, alpha-L-iduronosyl-2-sulphate(1----4)2,5-anhydro-D-[1-3H]-mannitol 6-sulphate and 4-methylumbelliferyl alpha-L-idoside did not undergo catalysis and were not inhibitory. A model of the catalytic requirements of alpha-L-iduronidase is proposed.

Catalysis↗
PubMed1983

Improved concanavalin A-Sepharose elution by specific readsorption of glycoproteins.

Elution of bound glycoproteins from concanavalin A-Sepharose can be made more efficient by their readsorption to a Blue A agarose column (specific) and Green A agarose column (less-specific) during recycling of the elution buffer. Three lysosomal enzymes were eluted in this way with marked improvement in their specific activities, time and handling and amount of eluting ligand used.

Adsorption↗
PubMed1983

Acetyl CoA:alpha-glucosaminide N-acetyl transferase: partial purification from human liver.

The lysosomal enzyme acetyl CoA:alpha-glucosaminide N-acetyltransferase (GNAT) was shown to be an integral membrane protein requiring high concentrations of the detergent Triton X-100 for maximal solubilization. Using a concentration dependent Triton X-100 solubilization procedure and Concanavalin A-Sepharose affinity chromatography, GNAT was purified 50-fold with a yield of 45%. GNAT activity was separated from N-acetyltransferase activity toward glucosamine 6-phosphate, an alternative non-lysosomal pathway for glucosamine metabolism. GNAT was different from other lysosomal enzymes which bound to Concanavalin A-Sepharose in that both alpha-methylmannoside and Triton X-100 were required for elution of enzyme activity. GNAT activity, which bound to Concanavalin A-Sepharose, required at least one other component which did not bind for maximal expression of enzyme activity and for storage stability. Phospholipids and glycolipids, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, sphingomyelin and gangliosides, and bovine serum albumin allowed expression of enzyme activity and storage stability similar to the component(s) which did not bind to Concanavalin A-Sepharose.

Acetyltransferases↗
PubMed1983

Cellular location of N-acetyltransfer activities toward glucosamine and glucosamine-6-phosphate in cultured human skin fibroblasts.

The intracellular location in normal human cultured skin fibroblasts of the N-acetyltransferase activities that transfer the acetyl group from acetyl-CoA to the 2-amino group of glucosamine and glucosamine-6-phosphate have been investigated. Organelles have been separated using a combination of differential centrifugation and free flow electrophoresis. The intracellular distribution of the enzyme involved in the N-acetyltransfer to glucosamine and an alpha-glucosaminide disaccharide indicated that this enzyme activity concentrates mainly with lysosomal organelles whereas the activity associated with N-acetyltransferase to glucosamine-6-phosphate is non-lysosomal. It is proposed that acetyl-CoA: alpha-glucosaminide N-acetyltransferase may be used as a convenient enzyme marker of lysosomal organelle membranes.

Acetyltransferases↗
PubMed1982

Irreversible inhibition of fatty acid synthase from rat mammary gland with S-(4-bromo-2,3-dioxobutyl)-CoA. Effect on the partial reactions, protection by substrates and stoichiometry studies.

Fatty acid synthase from lactating rat mammary gland is rapidly and irreversibly inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. Of the seven partial reactions catalysed by the enzyme, the inhibition of the overall catalytic activity is closely paralleled only by inhibition of the beta-oxoacyl synthase (condensing) partial reaction. Three partial reactions. Beta-oxoacyl reductase, beta-hydroxyacyl dehydratase and enoyl reductase, are inhibited to a modest degree. The three partial reactions known to involve an acyl-CoA/CoA-binding site, acetyl acyltransferase, malonyl acyltransferase and palmitoyl thioesterase, are not inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. The modification process does not cause the enzyme to dissociate into catalytically incompetent monomers. Stoichiometric studies suggest that approx. 6 mol of reagent are incorporated per mol of totally inhibited enzyme (dimer). The formation of acylated enzyme from either acetyl-CoA or malonyl-CoA protects the enzyme equally well against S-(4-bromo-2,3-dioxobutyl)-CoA. Also, pretreatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid), a thiol-specific reagent reported to block essential thiol groups in the condensing partial reaction, protects against inhibition by the reagent. On the other hand, the presence of up to 770 microM-S-acetonyl-CoA or dethio-CoA does not protect the enzyme from irreversible inhibition. Together, the results suggest that the primary inhibitory process is a bimolecular reaction resulting in alkylation of essential thiol groups in the condensing partial reaction: this process does not require the obligatory formation of a Michaelis-Menten complex of enzyme and reagent before the alkylation reaction.

Acyl Coenzyme A↗
PubMed1981

2-Bromoacetyl-SCoA.

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Acetyl Coenzyme A↗
PubMed1981

Insulin receptor synthesis and turnover in differentiating 3T3-L1 preadipocytes.

3T3-L1 "preadipocytes" can be induced to differentiate in culture into cells having the morphological and biochemical characteristics of adipocytes. The binding of 125I-insulin to the cell-surface of differentiated and undifferentiated 3T3-L1 cells and nondifferentiating 3T3-C2 cells was compared. In the absence of agents which induce adipocyte conversion, ie, insulin or insulin plus methylisobutylxanthine (MIX) and dexamethasone (DEX), 3T3-L1 cells fail to express the adipocyte phenotype and maintain a constant number of insulin binding sites. Induction of adipocyte conversion with 3T3-L1 cells in the presence of insulin causes apparent down-regulation of insulin receptors followed by a 12--15-fold increase in receptor number which parallels differentiation. Approximately 170,000 insulin binding sites per cell are expressed when greater than 75% of the cells have differentiated. The rise of insulin receptor level is differentiation-dependent. 3T3-C2 cells, which do not differentiate in the presence of insulin or insulin plus MIX and DEX, exhibit only insulin-induced down-regulation of insulin receptors. The increase of insulin receptor level in 3T3-L1 cells in receptor-specific since the levels of epidermal growth factor receptor or choleragen receptor, respectively, remain constant or decrease substantially. A heavy isotope, density-shift technique was used to analyze insulin receptor synthesis and turnover in cells labeled with "heavy" (2H, 13C, and 15N) amino acids. Solubilized newly-synthesized "heavy" and old "light" receptors were separated by isopycnic banding on CsCl gradients and quantitated. The size of the soluble receptor isolated after isopycnic banding in CsCl gradients is approximately 400,000 daltons. Mixing of "light" and "heavy" membranes prior to extraction of receptor revealed no change in "light" or "heavy" receptor isopycnic banding densities. Thus, no detectable interchange of subunits occurs between receptor molecules during extraction or equilibrium centrifugation. Insulin receptor synthesis and turnover, studied by the density-shift technique showed that the rise of receptor level during differentiation results primarily from an increased rate of receptor synthesis. The rate of insulin receptor degradation was not significantly altered. The t1/2 for degradation of the insulin receptor in differentiated 3T3-L1 cells in culture was 6--7 hours in the presence of insulin. Removal of insulin from the medium did not materially affect the rate of receptor degradation. Inhibition of protein synthesis with cycloheximide causes a lengthening of the t1/2 for insulin receptor degradation to 26 hours. Thus, the synthesis of a short-lived protein appears to be required for a critical step in the pathway of insulin receptor degradation.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Adaptive Behavior Scale, Part Two: predictive efficiency of severity and frequency scores.

Severity and frequency of occurrence methods of scoring the Adaptive Behavior Scale, Part Two, were compared using ratings for seven groups of mentally retarded persons. Comparisons involved separately correlating the two methods with independently obtained clinical impressions of symptomatology. The severity scoring system predicted approximately 11 percent more of the variance in the criterion than did frequency scores. These results provide considerable construct and predictive validity for the severity scoring system.

Adolescent↗
PubMed1980

Adaptive Behavior Scale part two: Relative severity of maladaptive behavior.

Part Two of the Adaptive Behavior Scale contains statements that cover 13 domains of maladaptive behavior. The present system of scoring and profiling reflects the frequency of occurrence of behavior (either occasionally or frequency) and not severity or relative importance. Thirty-three psychologists with experience in mental retardation rated the 452 statements contained in Part Two along a continuum of severity. Median severity scores were computed for all statements. Results indicated that statements prefaced by "frequently" were judged as more serious than those prefaced by "occasionally," and domains varied greatly with regard to relative severity. Correlational analysi revealed that irrespective of absolute differences due to frequency of occurrence, different types of maladaptive behavior were systematically placed along a relative continuum. Substantial reliability was found among judgments. Possible clinical and psychometric advantages of using these severity scores were suggested.

Humans↗
PubMed1976

Psychometric, diurnal, and electrophysiological correlates of activation.

Several studies were performed using Thayer's Activation-Deactivation Adjective Check List (AD-ACL). Factor analysis of college students' scores identified a single activation factor representing a continuum ranging from sleep through wakefulness. Next, changes over the diurnal cycle in activation factor scores were demonstrated. One-week test-retest reliability was also investigated. Finally, activation scores were related to pulse rate, respiration rate, and skin resistance level under passive and active conditions. Correlations between factor scores and electrophysiological measures were higher than were intercorrelations of electrophysiological measures, indicating that scores on this revised activation factor represent a valid measure of phenomenological bodily activation level.

Activity Cycles↗