Particulate Membrane Preparations Research Articles

Role of a mannosyl lipid intermediate in the synthesis of Neurospora crassa glycoproteins

Particulate membrane preparations from Neurospora crassa incorporated mannose from GDP-[14C] mannose into endogenous lipid and particulate protein acceptors. Synthesis of the mannosyl lipid is reversible in the presence of GDP. Chemical and chromatographic characterization of the mannosyl lipid suggest that it is a mannosylphosphorylpolyisoprenol. The other endogenous acceptor was precipitated by trichloracetic acid. Gel filtration and electrophoresis studies before and after treatment with proteolytic enzymes indicate that the second acceptor is a glycoprotein(s). beta Elimination studies on the mannosyl protein formed from GDP-[14C] mannose with Mg2+ in the reaction mixture or formed from mannosyl lipid indicate thad with the peptide chain. Several lines of evidence indicate that in Neurospora crassa the mannosyl lipid is an obligatory intermediate in the in vitro mannosylation of the protein. (a) At 15 degrees C the initial formation of the mannosyl lipid is faster than the initial formation of the mannosyl protein. (b) Exogenous partially purified mannosyl lipid can function as a mannosyl donor for the synthesis of the mannosyl protein. This reaction was also dependent on a divalent metal. The rate of this reaction was optimal at a concentration of Triton X-100 which effectively inhibited the transfer of mannose from GDP-[14C] mannose to lipid and protein, indicating that GDP-mannose was not an intermediate in the transfer of mannose from lipid to protein. The mannosyl protein formed in this reaction was indistinguishable by several criteria from the mannosyl protein formed from GDP-[14C] mannose and Mg2+. (c) The effect of a chase with an excess of unlabeled GDP-mannose on the incorporation of mannose into endogenous acceptors was immediate cessation of the synthesis and subsequent turnover of the mannosyl lipid; in contrast, however, incorporation of mannose into protein continued and was proportional to the loss of mannose from the mannosyl lipid.

Synthesis of Yeast Wall Glucan

Saccharomyces cerevisiae was treated with a mixture of toluene and ethanol to make it permeable to small molecules. This treatment unmasked a glucan synthetase activity which was assayed with UDP-[U-14C]glucose. About 60% of the polymer formed was beta-(I leads to 3)glucan. No labelled lipids were detected. The 14C incorporated was recovered in a particulate membrane preparation isolated by differential centrifugation. When the particles themselves were assayed for glucosyl transfer activity none was found. The toluene-treated preparations also catalysed the transfer of mannosyl residues from GDP-mannose to polymeric materials by a process independent of glucosyl transfer.

Bovine adrenal cortex adenylate cyclase: Properties of the particulate enzyme and effects of guanyl nucleotides

The preparation of a partially purified plasma membrane fraction from bovine adrenal cortex is described. Adenylate cyclase in this particulate preparation retained high sensitivity to ACTH and is also stimulated by 5'-guanylyl-imidodiphosphate [Gpp(NH)p]. GTP, in contrast to Gpp(NH)p, had very little intrinsic activity to stimulate activity to stimulate adenylate cyclase. GTP could however, with high affinity, inhibit the Gpp(NH)p effects on adenylate cyclase. When the concentration of creatine phosphate, a component of the ATP-regenerating system in the adenylate cyclase assay mixture, was lowered from 20 to 2 mM (at 0.1 mM ATP, 5 MM MG2+) GTP, dGTP and other nucleotides like ITP and much less UTP or CTP gained considerable intrinsic activity in the presence of ACTH to stimulate adenylate cyclase. The apparent affinities of the nucleotides for ACTH-stimulated adenylate cyclase from bovine adrenal cortex (at 2 mM creatine phosphate) were, GTP = dGTP greater than Gpp(NH)p greater than Gpp(CH2)p (5'-guanylyl-beta, gamma-methylene-diphosphonate) greater than ITP greater than UTP greater than CTP. These findings indicate that regulatory nucleotide binding sites exist for bovine adrenal cortex adenylate cyclase. Their specificity is similar to the nucleotide sites modulating angiotensin binding in bovine adrenal cortex plasma membranes (Glossmann et al., 1974a). The regulatory nucleotide binding sites for the adrenal cortex adenylate cyclase complex can also be identified under conditions where only Gpp(NH)p has high intrinsic activity (e.g. at 20 mM creatine phosphate) but other nucleotides like GTP act as antagonists. Both stimulants, ACTH and Gpp(NH)p, appear to remain firmly bound to the particulate membrane preparation, as suggested by preincubation experiments.

Bromacetyl-oxytocin, an irreversible inhibitor of neurohypophyseal hormone-stimulated adenylate cyclase, and a possible affinity label for hormone receptors.

The synthesis of bromoacetyl—oxytocin, a neurohypophyseal hormone analog containing a chemically reactive group, is described. Bromoacetyl—oxytocin fails to enhance adenylate cyclase activity in particulate membrane preparations of rabbit kidney and toad bladder. Preincubation with this analog inhibits the stimulatory effects of neurohypophyseal hormones upon cyclic 3′,5′–AMP formation in both cyclase preparations; the inhibition is dependent on the duration and temperature of preincubation as well as on the concentration of bromoacetyl—oxytocin. The results of several types of experiments indicate that bromoacetyl—oxytocin acts as a specific irreversible inhibitor of neurohypophyseal hormone action. The findings in this study suggest that this analog, once radioactively labeled, may be useful as an affinity label for the isolation of neurohypophyseal hormone receptor molecules. (Endocrinology91: 39, 1972)

Biosynthesis of the wall teichoic acid in Bacillus licheniformis.

1. The biosynthesis of the wall teichoic acid, poly(glycerol phosphate glucose), has been studied with a particulate membrane preparation from Bacillus licheniformis A.T.C.C. 9945. The precursor CDP-glycerol supplies glycerol phosphate residues, whereas UDP-glucose supplies only glucose to the repeating structure of the polymer. 2. Synthesis proceeds through polyprenol phosphate derivatives, and chemical studies and pulse-labelling techniques show that the first intermediate is the phosphodiester, glucose polyprenol monophosphate. CDP-glycerol donates a glycerol phosphate residue to this to give a second intermediate, (glycerol phosphate glucose phosphate) polyprenol. 3. The glucose residue in the lipid intermediates has the beta configuration, and chain extension in the synthesis of polymer occurs by transglycosylation with inversion of anomeric configuration at two stages.