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Biomedical subjects

R M Bell

Publications and source records attributed to R M Bell.

At least 253 records · Page 14Linked to original sources
PubMed1977

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.

Adenosine Triphosphate↗
PubMed1976

Triacylglycerol synthesis in isolated fat cells. Evidence that the sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities are dual catalytic functions of a single microsomal enzyme.

The acyl-CoA:sn-glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (glycerol-P acyltransferase) and acyl-CoA:dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42) (DHAP acyltransferase) activities were investigated in vitro in order to evaluate the quantitative contribution of the glycerol-P and DHAP pathways for the synthesis of triacylglycerols in isolated fat cells and to test the hypothesis that these two activities may be dual catalytic functions of a single enzyme. More than 85% of both acyltransferase activities was associated with the microsomal subcellular fraction. The microsomal glycerol-P acyltransferase activity showed an apparent Km of 8 muM for glycerol-P with a Vmax of 15.6 nmol/min/mg, while the DHAP acyltransferase activity showed an apparent Km of 40 muM for DHAP with a Vmax of 9.7 nmol/min/mg. Glycerol-P was a competitive inhibitor (Ki = 7.2 muM) of the DHAP acyltransferase, and DHAP was a competitive inhibitor (Ki = 92 muM) of the glycerol-P acyltransferase. The two acyltransferase activities showed virtual identity in their pH dependence, acyl-CoA chain length dependence, thermolability, and inactivation by N-ethylmaleimide. Trypsin, detergents, collagenase, phospholipases, and various salts and organic solvents also had similar effects on both activities. Taken as a whole, the data strongly suggest that the microsomal glycerol-P and DHAP acyltransferase activities actually represent dual functions of a single enzyme. Calculations based on the above kinetic constants and previously reported glycerol-P and DHAP pools in adipocytes suggest that the in vivo ratio of glycerol-P to DHAP acylation should be greater than 24:1.

Acyltransferases↗
PubMed1976

Triacylglycerol synthesis in isolated fat cells. Studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerols.

The acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) activity of isolated fat cells was predominantly (89%) localized to the microsomal subcellular fraction by assays based on the conversion of 1,2-[3H]diacyl-sn-glycerol to triacylglycerol using 1 to 4 mug of protein. A complementary assay based on the conversion of [3H]palmitoyl-CoA to triacylglycerol was developed. These methods, 100 to 1000 times more sensitive than those previously employed, were used to characterize the microsomal activity. The choice of dispersing agent for addition of diacylglycerol to the reaction mixture was crucial. Addition of diacylglycerol in ethanol resulted in the highest diacylglycerol acyltransferase activity of the methods tested. Tween 20, which has previously been employed as the dispersing agent, severely inhibited the activity. A broad pH optimum from 7.4 to 8.0 was noted and several salts stimulated the activity more than 2-fold. The activity was unstable at temperatures of 28 degrees and above. Dependences on acyl-CoAs containing 6 to 18 carbon atoms were investigated using bacterial diacylglycerol. Acetyl- and butyryl-CoA were not substrates. Highest diacylglycerol acyltransferase activities were observed with decanoyl-CoA and lower activities were noted with longer and shorter saturated chains. Maximal activity with oleoyl-CoA was only 34% of that seen with stearoyl-CoA. No simple relationship between the critical micellar concentrations of the acyl-CoAs employed and diacylglycerol acyltransferase activity was observed. The dependences on diacylglycerols containing fatty acids 6 to 18 carbon atoms in length were investigated with [3H]palmitoyl-CoA. While all the 1,2-diacyl-sn-glycerols tested were substrates, diacylglycerol acyltransferase activity was highest with 1,2-dioleoylglycerol. Maximum activity with the bacterial diacylglycerol was 86% that with 1,2-dioleoylglycerol. The diacylglycerol concentrations required for half-maximal velocity were 20 to 40 muM for long chain diacylglycerols and 2 to 3 muM for short chain diacylglycerols; these were 75- to 750-fold lower than previously reported. Microsomal diacylglycerol acyltransferase specific activities from isolated cells around 50 nmol/min/mg, 10- to 50-fold higher than previously reported from adipose tissue, were typical using dioleoylglycerol and palmitoyl-CoA as substrates. Diacylglycerol acyltransferase specific activities were 17-fold higher in microsomes from isolated fat cells than any other tissue examined. The diacylglycerol acyltransferase appears to have specificity with respect to acyl-CoAs and diacylglycerols, but a definitive interpretation is limited by the lack of data on the physical properties of these substrates in solution under the conditions employed.

Acyltransferases↗
PubMed1976

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.

Adenosine Triphosphate↗
PubMed1975

A mutant of Escherichia coli auxotrophic for organic phosphates: evidence for two defects in inorganic phosphate transport.

An inorganic phosphate transport mutant has been isolated as a sn-glycerol-3-phosphate auxotroph and characterized genetically. Two lesions are responsible for the transport defect. One lesion, pst, is located at minute 74 of the E. coli genetic map while the other lesion, pit, is located at minute 68. All "K10" strains that were examined carry the pit lesion. Evidence is presented that the glycerol phosphate and hexose phosphate transport systems are not important inorganic phophate transport systems. The mapping data indicate that the genetic distance between malA and xyl is greater than that now allowed.

Bacterial Proteins↗
PubMed1975

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Effect of cessation of net phospholipid synthesis on cytoplasmic and outer membranes.

The effect of cessation of net phospholipid synthesis on the cytoplasmic and outer membranes of Escherichia coli was investigated in a mutant strain defective in the first enzyme of phospholipid synthesis, the sn-glycerol-3-phosphate (glycerol-P) acyltransferase. The glycerol-P (glycerol) auxotropic phenotype of this strain resulted from an altered membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of the parental activity. When net phospholipid synthesis was halted during glycerol deprivation, both soluble and cell envelope protein synthesis continued. Fractionation of the membranes derived from glycerol-supplemented and glycerol-deprived cultures by isopycnic banding in sucrose gradients revealed that both the cytoplasmic and outer membranes of the deprived culture banded at higher buoyant densities. The protein/phospholipid ratio of both the cytoplasmic and outer membranes increased approximately 60% during the period of glycerol deprivation. The distribution of two cytoplasmic membrane activities, NADH oxidase and 1-acylglycerol-P acyltransferase, and an outer membrane activity, phospholipase A1, showed that the total membranes derived from glycerol-deprived cultures were separated cleanly into cytoplasmic and outer membrane fractions. Both cytoplasmic and outer membrane proteins were synthesized and integrated into their respective membranous structures when net phospholipid synthesis was halted. Hence, the biosynthesis of membrane phospholipid and membrane protein are not tightly coupled. Further, these data suggest that cellular control mechanisms exist which maintain the protein content of both membranous structures below the point where they are saturated with protein.

Bacterial Proteins↗
PubMed1975

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.

Acyltransferases↗
PubMed1975

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Phenotypic suppression of sn-glycerol-3-phosphate acyltransferase Km mutants by loss of feedback inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Revertants of Escherichia coli mutants defective in the first enzyme of membrane phospholipid synthesis, sn-glycerol-3-phosphate (glycerol-P) acyltransferase, were investigated. These glycerol-P acyltransferase mutants, selected as glycerol-P auxotrophs, contained membranous glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than the parental activity. The glycerol-P acyltransferase activity was also more thermolabile in vitro than the parental activity. Most revertants no longer requiring glycerol-P for growth regained glycerol-P acyltransferase activity of normal thermolability and apparent Km for glycerol-P. However, two novel revertants were isolated which retained an abnormal glycerol-P acyltransferase activity. The glycerol-P dehydrogenase activities of these novel revertants were about 20-fold less sensitive to feedback inhibition by glycerol-P. The feedback-resistant glycerol-P dehydrogenase co-transduced with gpsA, the structural gene for the glycerol-P dehydrogenase. Further transduction experiments demonstrated that the feedback resistant glycerol-P dehydrogenase phenotypically suppressed the glycerol-P acyltransferase Km lesion. The existence of the class of glycerol-P auxotrophs which owe their phenotype to the glycerol-P acyltransferase Km lesion therefore depends on the feedback regulation of glycerol-P synthesis in E. coli.

Acyltransferases↗
PubMed1975

Solitary choroidal metastasis from bronchial carcinoid.

This report describes the clinical, light microscopical, and electron microscopical features of a metastasis from a bronchial carcinoid tumour occurring in a 29-year-old white woman. The eye lesions was diagnosed 30months after resection of the primary pulmonary tumour. Ophthalmoscopically the patient was observed to have a solid choroidal mass. Enucleation was carried out because of the possibility that the tumour was a primary choroidal melanoma. Enucleation was also indicated because of the relatively good prognosis for long-term survival in patients following excision of metastases from a bronchial carcinoid tumour. By light microscopy the metastasis was seen to be composed of cords; and ribbons of cells which showed positive staining characteristics for argentophilia. On electron microscopical study, neurosecretory vesicles, numerous microvilli, mitochondria, and light and dark cells, characteristic of endocrine tissue in different states of activity were noted.

Adult↗
PubMed1975

Millipore filter assay for long-chain fatty acid:CoASH ligase activity using 3H-labeled coenzyme A.

A novel radiochemical assay for long-chain fatty acid:CoASH ligase activity (AMP) (EC 6.2.1.3) has been developed based on the conversion of [3H]CoASH to long-chain fatty acyl CoA. Fatty acyl [3H]CoA was quantitatively retained on Millipore filters upon filtration of the acidified reaction mixture under conditions where the [3H]CoASH was not retained. The assay was developed using microsomes derived from isolated fat cells as the source of fatty acid:CoASH ligase activity. The assay performed at 25 degrees C for 10 min was linear with added microsomal protein up to 7 mug. The assay was linear with time up to 24 min when 1 mug of protein was employed. Fatty acid:CoASH ligase activity was strongly dependent on ATP and magnesium, was stimulated by Triton WR-1339, and was two- to fivefold dependent on added fatty acid. The filter assay is easier than existing assays based on incorporation of labeled fatty acid and is equally sensitive.

Animals↗
PubMed1974

Mutants of Escherichia coli defective in membrane phospholipid synthesis: macromolecular synthesis in an sn-glycerol 3-phosphate acyltransferase Km mutant.

sn-Glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli have been selected from a strain which cannot aerobically catabolize G3P. The auxotrophy resulted from loss of the biosynthetic G3P dehydrogenase (EC 1.1.1.8) or from a defective membranous G3P acyltransferase. The apparent K(m) of the acyltransferase for G3P was 11- to 14-fold higher (from about 90 mum to 1,000 to 1,250 mum) in membrane preparations from the mutants than those of the parent. All extracts prepared from revertants of the G3P dehydrogenase mutants showed G3P dehydrogenase activity, but most contained less than 10% of the wild-type level. Membrane preparations from revertants of the acyltransferase mutants had apparent K(m)'s for G3P similar to that of the parent. Strains have been derived in which the G3P requirement can be satisfied with glycerol in the presence of glucose, presumably because the glycerol kinase was desensitized to inhibition by fructose 1,6-diphosphate. Investigations on the growth and macromolecular synthesis in a G3P acyltransferase K(m) mutant revealed that upon glycerol deprivation, net phospholipid synthesis stopped immediately; growth continued for about one doubling; net ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein nearly doubled paralleling the growth curve; the rate of phospholipid synthesis assessed by labeling cells with (32)P-phosphate, (14)C-acetate, or (3)H-serine was reduced greater than 90%; the rates of RNA and DNA synthesis increased as the cells grew and then decreased as the cells stopped growing; the rate of protein synthesis showed no increase and declined more slowly than the rates of RNA and DNA synthesis when the cells stopped growing. The cells retained and gained in the capacity to synthesize phospholipids upon glycerol deprivation. These data indicate that net phospholipid synthesis is not required for continued macromolecular synthesis for about one doubling, and that the rates of these processes are not coupled during this time period.

Acetates↗
PubMed1974

Mutants of Escherichia coli defective in membrane phospholipid synthesis: mapping of the structural gene for L-glycerol 3-phosphate dehydrogenase.

The structural gene for the biosynthetic l-glycerol 3-phosphate dehydrogenase has been mapped at min 71.5 on the Escherichia coli chromosome. This gene (gpsA) is co-transduced with the xyl, mtl, and pyrE loci. Three-factor conjugational crosses and the transduction data indicate that the order of loci in this region of the chromosone is mtl, gltE, gpsA, gadR, gadS, pyrE. Study of a temperature-sensitive gpsA mutant possessing a dehydrogenase of increased thermolability indicated that gpsA is the structural gene for the dehydrogenase. All dehydrogenase-deficient strains tested were mapped very close to the gpsA locus. Attempts at genetic complementation analysis were unsuccessful.

Cell Membrane↗