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Biomedical subjects

R M Bell

Publications and source records attributed to R M Bell.

At least 163 records · Page 9Linked to original sources
PubMed1986

Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli.

Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function. Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive. Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification. Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping. Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined. Unexpectedly, disproportionalities occurred during the purification. However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide. This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E. coli strain and from plasmid-containing strains which express the plsB gene product to different extents. The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional. In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution. The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.

Acyltransferases↗
PubMed1986

Cloning and expression of multiple protein kinase C cDNAs.

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.

Amino Acid Sequence↗
PubMed1986

Crystalline arrays of the Escherichia coli sn-glycerol-3-phosphate acyltransferase, an integral membrane protein.

The gene encoding the Escherichia coli sn-glycerol-3-phosphate acyltransferase, plsB, was inserted into hybrid plasmids under transcriptional control of the lambda PL and tac promoters. Enzymatic activities 35-50-fold above wild type and a large increase in glycerol-P acyltransferase polypeptide were obtained. Thin section electron microscopy of the cells overproducing the glycerol-P acyltransferase revealed 235-245-A diameter tubular structures associated with the cytoplasmic membrane. These structures were released from the cell by osmotic lysis and purified on Matrex Gel Green A. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the dominant protein constituent of the tubules was the glycerol-P acyltransferase. Analysis of tubule-enriched fractions isolated by differential centrifugation revealed a decreased phospholipid to protein ratio as compared to total and cytoplasmic membrane fractions. At high magnification, negative stained tubules displayed ordered arrays of stain-excluding components projecting 50-60 A from the cytoplasmic surface. Optical diffraction patterns from the micrographs contained intense layer lines at (1/78 A) and (1/39 A) along the tubule axis and a prominent spot at (1/62 A) near the equator. From compositional and structural data, 18-37% of the polypeptide volume is estimated to lie within the hydrophobic domain of the tubule membrane.

Acyltransferases↗
PubMed1986

Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine.

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.

Animals↗
PubMed1986

Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase. Simultaneous loss of dihydroxyacetone phosphate acyltransferase indicates a common gene.

Fourteen independent mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase activity were isolated using a colony autoradiographic screening technique. All 14 mutants were similarly defective in dihydroxyacetone phosphate acyltransferase activity. The mutations were recessive and fell into a single complementation group. Tetrad analysis gave results consistent with mutations in a single nuclear gene affecting both activities. sn-Glycerol-3-phosphate acyltransferase activity from different mutant strains exhibited different substrate dependencies and differing responses to temperature, detergent, and pH. In each case, the response of the dihydroxyacetone phosphate acyltransferase activity was similar to that of the sn-glycerol-3-phosphate acyltransferase. These results are consistent with the mutations occurring in the structural gene. The data also establish that the predominant dihydroxyacetone phosphate acyltransferase activity in yeast is a second activity of the sn-glycerol-3-phosphate acyltransferase.

Acyltransferases↗
PubMed1986

Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells.

A simple enzymatic method for the quantitation of the mass of sn-1,2-diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products. The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to [32P]phosphatidic acid. The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol. The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements. The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid. In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively. When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature. These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C. Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation.

Animals↗
PubMed1986

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols.

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.

Binding, Competitive↗
PubMed1986

Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies.

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS), and sn-1,2-dioleoylglycerol (diC18:1) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC18:1. Activity of protein kinase C was dependent on PS, diC18:1, and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC18:1 mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC18:1 rapidly equilibrate among the mixed micelles. At saturating calcium, the diC18:1 dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC18:1 gave maximal activity whereas 6 mol % PS and 6 mol % diC18:1 did not give maximal activity. diC18:1 dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC18:1/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC18:1 increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all the phospholipids tested in the mixed micelle assay. diC18:1 was found to modulate the amount of calcium required for maximal activity. As the level of Ca2+ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.

Adenosine Triphosphate↗
PubMed1986

Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets.

Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.

Blood Platelets↗
PubMed1986

sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence.

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.

Adenosine Triphosphate↗
PubMed1986

Thyroid carcinoma.

The issue of appropriate management for thyroid neoplasms remains unsettled. Confusion concerning histology and classification, controversy as to appropriate therapy, the relative indolence of most differentiated thyroid tumors, and various levels of surgical skill have contributed to the prejudice of various approaches to the management of such neoplasms. It appears unlikely that the controversy will be resolved in the immediate future. If total thyroidectomy can be performed by a skilled surgeon with minimal complications, this approach seems theoretically justifiable for the management of most differentiated tumors, facilitating follow-up postoperatively. Conclusive evidence for improved survival with this rationale is insufficient.

Adenocarcinoma↗
PubMed1986

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C.

Animals↗
PubMed1986

Activation of protein kinase C inhibits synthesis and release of decidual prolactin.

Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidua, we have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanoylglycerol (diC8), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% (P less than 0.01) at 100 microM. Diolein (100 microM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3% (P less than 0.05). Distearin and dipalmitin, which are much less effective in activating protein kinase C, and monopalmitin and tripalmitin, which do not activate protein kinase C, had no significant effect on prolactin release. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4 beta-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, while the protein kinase C inactivate 4 alpha-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. The amounts of prolactin synthesized by cells exposed to diC8 (300 microM) or PMA (10(-7) M) for 30 min were 56.3 and 50.0% less than that synthesized by control cells (P less than 0.01 in each instance).(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1986

Sn-1,2-diacylglycerols and phorbol esters stimulate the synthesis and release of human placental lactogen from placental cells: a role for protein kinase C.

Activation of calcium-stimulated phospholipid-dependent protein kinase C (PKC) by diacylglycerols and phorbol esters has been shown to mediate the release of secretory proteins in several systems. To determine whether PKC activation is involved in regulation of the release of human placental lactogen (hPL) from the placenta, we examined the effects of various acylglycerols and phorbol esters on the release of hPL from cultured human trophoblast cells. Sn-1,2-dioctanoylglycerol (diC8) and phorbol-12-myristate-13-acetate (PMA), both of which stimulate placental protein kinase C activity, caused dose-dependent increases in hPL release over a 0.5-h period. The maximal amounts of hPL released in response to diC8 (300 microM) and PMA (10(-8) M) were 200-300% and 150-225% greater, respectively, than that released in response to diluent alone. Acylglycerols and phorbol esters, which are less potent stimulators of PKC activity in other systems, stimulated hPL release to a lesser extent than either diC8 or PMA. PKC-inactive acylglycerols and phorbol esters were without effect. After 0.5 h of exposure, diC8 (300 microM)- and PMA (10(-8) M)-exposed cells synthesized 257.5% and 250.3% more hPL than control cells. Cycloheximide at a dose (50 micrograms/ml) that inhibited the synthesis of trichloroacetic acid-precipitable [35S]methionyl placental proteins by more than 80% completely blocked the stimulatory effects of diC8 and PMA on hPL synthesis and release. Although diC8 and PMA stimulated the synthesis and release of hPL, these compounds had no effect on the release of hCG and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. The demonstration that acylglycerols and phorbol esters stimulate the synthesis and release of hPL strongly implicates protein kinase C activation in the mechanisms of hPL synthesis and release.

Cycloheximide↗