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Biomedical subjects

M D Lane

Publications and source records attributed to M D Lane.

At least 181 records · Page 10Linked to original sources
PubMed1978

Mechanism of action of ribulose bisphosphate carboxylase/oxygenase.

RuBP carboxylase-oxygenase appears to catalyze carboxylation and oxygenation by homologous mechanisms. A common binding site exists on the enzyme for the acceptor substrate, RuBP. A mechanism is proposed whereby RuBP is isomerized, and a carbanion is generated at C2. Then, either CO2 or O2 is added as an electrophile at C2 to form the corresponding 3-keto-2-carboxy-RBP or 3-keto-2-hydroperoxy-RBP adduct. Hydrolytic cleavage at the C2-C3 bonds of these intermediates by the enzyme is envisioned to produce 2 molecules of 3-phosphoglycerate in the carboxylation sequence and 1 molecule of phosphoglycolate and 1 molecule of 3-phosphoglycerate in the oxygenation sequence. Further work will be necessary to establish the validity of the proposed mechanism.

Carbon Dioxide↗
PubMed1977

Changes in mammary-gland acetyl-coenzyme A carboxylase associated with lactogenic differentiation.

The process leading to the rise of acetyl-CoA carboxylase activity in rat mammary tissue after the onset of lactation was investigated. The kinetics of change in enzyme activity and enzyme immunotitratable with antibody against avian liver acetyl-CoA carboxylase were determined during the course of lactogenic differentiation. The antibody inactivates and specifically precipitates acetyl-CoA carboxylase from rat mammary tissue as well as that from chicken liver cytosol. Characterization of the immunoprecipitate of the mammary tissue carboxylase by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals a single biotin-containing polypeptide of about 230000mol.wt. This molecular weight is approximately twice that reported for the avian liver enzyme. However, chicken liver cytosol prepared in the presence of trypsin inhibitor and subjected to immunoprecipitation gives rise to a biotin-containing subunit of 230000mol.wt. as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; omission of proteinase inhibitor leads to a subunit(s) approximately one-half this size. Throughout gestation both carboxylase activity and amounts of immunotitratable enzyme remained low; however, after parturition both parameters rose concomitantly to values 30-40 times the initial values. Therefore the elevated concentration of acetyl-CoA carboxylase appears to result from an increased rate of synthesis of enzyme relative to degradation rather than to activation of a pre-existing form of the enzyme.

Acetyl-CoA Carboxylase↗
PubMed1977

3-Hydroxy-3-methylgutaryl-CoA synthase. Participation of acetyl-S-enzyme and enzyme-S-hydroxymethylgutaryl-SCoA intermediates in the reaction.

Acetyl-CoA reacts stoichiometrically with a cysteinyl sufhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme (Miziorko H.M., Clinkenbeard, K.D., Reed, W.D., and Lane, M.D. (1975) J. Biol. Chem. 250, 5768-5773). Evidence that acetyl-S-enzyme condenses with the second substrate, acetoacetyl CoA, to form enzyme-S-HMG-SCoA has been obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25 degrees with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]aceto-acetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA had been covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.

Acetates↗
PubMed1977

Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes.

Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in 125I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which greater than 75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40;000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant.

Adipose Tissue↗
PubMed1977

Mechanism for acute control of fatty acid synthesis by glucagon and 3':5'-cyclic AMP in the liver cell.

Labeling experiments with chicken liver cell monolayers and suspensions show that glucagon and N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) block fatty acid synthesis from acetate without appreciably affecting cholesterogenesis from acetate or acylglyceride synthesis from palmitate. Neither acetyl-CoA carboxylase [acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] activity assayed in the presence of citrate nor fatty acid synthetase activity is decreased in extracts of cells treated with glucagon. However, the cytoplasmic concentration of citrate, a required allosteric activator of acetyl-CoA carboxylase, is depressed more than 90% by glucagon or dibutyrl cyclic AMP. Pyruvate or lactate largely prevents the inhibitory action of these effectors on fatty acid synthesis by causing a large increase in cytoplasmic citrate level. Thus, it appears that glucagon, acting via cyclic AMP, inhibits fatty acid synthesis by blocking the formation of citrate, an essential activator of acetyl-CoA carboxylase.

Acetates↗
PubMed1977

Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects.

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.

Bucladesine↗
PubMed1976

Induction of lipogenesis during differentiation in a "preadipocyte" cell line.

3T3-L1 fibroblasts differentiate in culture into cells having adipocyte character. This transition is accompanied by a 40- to 50-fold rise in the incorporation of [14C]acetate into triglyceride. The increase in lipogenic rate is exactly parallel to a coordinate rise in the activities of the key enzymes of the fatty acid biosynthetic pathway (ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase). Immunological studies indicate that the elevated acetyl-CoA carboxylase activity is the product of an increased cellular enzyme level.

ATP Citrate (pro-S)-Lyase↗
PubMed1975

3-Hydroxy-3-methylglutaryl coenzyme A synthase. Evidence for an acetyl-S-enzyme intermediate and identification of a cysteinyl sulfhydryl as the site of acetylation.

Homogeneous liver 3-hydroxy-3-methylglutaryl coenzyme A synthase, which catalyzes the condensation of acetyl-CoA with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA, also carries out: (a) a rapid transacetylation from acetyl-CoA to 31-dephospho-CoA and (b) a slow hydrolysis of acetyl-CoA to acetate and CoA. Transacetylation and hydrolysis occur at 50 and 1 percent, respectively, the rate of the synthasecatalyzed condensation reaction. It appears that an acetyl-enzyme intermediate is involved in the transacetylase and hydrolase reactions of 3-hydroxy-3-methylglutaryl-CoA synthase, as well as in the over-all condensation process. Covalent binding to the enzyme of a [14C]acetyl group contributed by [1(-14)C]acetyl-CoA is indicated by migration of the [14C]acetyl group with the dissociated synthase upon electrophoresis in dodecyl sulfate-urea and by precipitation of [14C]acetyl-enzyme with trichloroacetic acid. At 0 degrees and a saturating level of acetyl-CoA, the synthase is rapidly (less than 20 s) acetylated yielding 0.6 acetyl group/enzyme dimer. Performic acid oxidation completely deacetylates the enzyme, suggesting the site of acetylation to be a cysteinyl sulfhydryl group. Proteolytic digestion of [14C]acetyl-S-enzyme under conditions favorable for intramolecular S to N acetyl group transfer quantitatively liberates a labeled derivative with a [14C]acetyl group stable to performic acid oxidation. The labeled oxidation product is identified as N-[14C]acetylcysteic acid, thus demonstrating a cysteinyl sulfhydryl group as the original site of acetylation. The ability of the acetylated enzyme, upon addition of acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA indicates that the acetylated cysteine residue is at the catalytic site.

Acetylation↗
PubMed1975

Intracellular localization of the 3-hydroxy-3-methylglutaryl coenzme A cycle enzymes in liver. Separate cytoplasmic and mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A generating systems for cholesterogenesis and ketogenesis.

Acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme synthase which comprise the 3-hydroxy-3-methylglutaryl-CoA-generating system(s) for hepatic cholesterogenesis and ketogenesis exhibit dual mitochondrial and cytoplasmic localization. Twenty to forty per cent of the thiolase and synthase of avian and rat liver are localized in the cytoplasmic compartment, the remainder residing in the mitochondria. In contrast, 3-hydroxy-3 methylglutaryl-CoA lyase, an enzyme unique to the "3-hydroxy-3-methylglutaryl-CoA cycle" of ketogenesis, appears to be localized in the mitochondrion. The small proportion, 4 to 8 percent, of this enzyme found in the cytoplasmic fraction appears to arise via leakage from the mitochondria during cell fractionation in that its properties, pI and stability, are identical to those of the mitochondrial lyase. These results are consistent with the view that ketogenesis which involves all three enzymes, acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA lyase, occurs exclusively in the mitochondrion, whereas cholesterogenesis, a pathway which involves only the 3-hydroxy-3-methylglutaryl-CoA synthesizing enzymes, is restricted to the cytoplasm. Further fractionation of isolated mitochondria from chicken and rat liver showed that all three of the 3-hydroxy-3-methylglutaryl-CoA cycle enzymes are soluble and are localized within the matrix compartment of the mitochondrion. Likewise, cytoplasmic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-CoA synthase are soluble cytosolic enzymes, no thiolase or synthase activity being detectable in the microsomal fraction. Chicken liver mitochondrial 3-hydroxy-3methylglutaryl-CoA synthase activity consists of a single enzymic species with a pI of 7.2, whereas the cytoplasmic activity is composed of at least two species with pI values of 4.8 and 6.7. Thus it is evident that the mitochondrial and cytoplasmic species are molecularly distinct as has been shown to be the case for the mitochondrial and cytoplasmic acetoacetyl-CoA thiolases from avian liver (Clinkenbeard, K. D., Sugiyama, T., Moss, J., Reed, W. D., and Lane, M. D. (1973) J. Biol. Chem. 248, 2275). Substantial mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase activity is present in all tissues surveyed, while only liver and kidney possess significant mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Therefore, it is proposed that tissues other than liver and kidney are unable to generate acetoacetate because they lack the mitochondrial synthase.

Adenosine Monophosphate↗
PubMed1975

Molecular and catalytic properties of mitochondrial (ketogenic) 3-hydroxy-3-methylglutaryl coenzyme A synthase of liver.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase has been purified to homogeneity from avian liver. The enzyme in dilute phosphate buffer, pH 7.0, has an S20,w of 5.7 S and a molecular weight of 105,000 determined by sedimentation equilibrium; the presence of 0.1 M KCl causes dissociation to a form one-half that size, i.e. about 57,000 daltons. Since the subunit molecular weight of the synthase determined by the dodecyl sulfate acrylamide gel method is 53,000, it appears that the native enzyme is a dimer composed of weight-homogeneous subunits. A number of molecular and catalytic properties allow the mitochondrial and cytoplasmic 3-hydroxy-3-methylglutaryl-CoA synthases to be distinguished. The pI of the homogeneous mitochondrial enzyme is 7.2. This value, while identical to that of the single isoelectric-focusing species of broken mitochondrial preparations, differs from those of the multiple 3-hydroxy-3-methylglutaryl-CoA synthases found in the cytoplasmic fraction which exhibit pI values of 4.8 and 6.7. Rabbit antibodies against the purified mitochondrial synthase are capable of precipitating the mitochondrial, but not the cytoplasmic, synthase(s) of avian liver. Finally, the synthase differ kinetically in their responses to divalent magnesium ion, the mitochondrial enzyme being inhibited and the cytoplasmic enzyme(s) activated. It is proposed that the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase of liver functions in ketogenesis while its cytoplasmic counterpart participates in cholesterogenesis (Clinken-Beard, K. D., Sugiyama, T., Reed, W. D. and Lane, M.D. (1975) J. Biol. Chem. 250, 3124-3134).

Animals↗