Canadian journal of biochemistry最新文献

The degradation of bis(monoacylglycero)phosphate by subcellular fractions of rat liver, using substrates labelled biosynthetically with [14C]oleic acid and chemically by catalytic exchange with tritium, was studied. Liver homogenates catalyzed maximum degradation at alkaline pH and subcellular fractionation localized this activity to microsomes. The degradation by microsomes was found to be a deacylation to lysophosphatidylglycerol and was without phosphodiesterase activity. The deacylation was maximal at pH 8.3 and did not require Ca2+ or Mg2+ but was stimulated by ethylenediaminetetraacetic acid and inhibited by Fe2+ and Hg2+. It was also inhibited by p-chloromercuribenzoate, deoxycholate, Triton X-100, and Triton WR-1339. The apparent Km was determined to be 5.5 X 10(-5) M and the corresponding V max was 4.1 nmol product released/min per milligram protein. The three labelled substrates were degraded by microsomes to give the same products in similar relative proportions. Degradation of bis(monoacylglycero)phosphate by lysosomes was maximal at acid pH as previously described by Y. Matsuzawa and K. Y. Hosteler. Contrary to their finding, deacylase activity in lysosomes was much greater than phosphodiesterase activity. The lysosomal deacylase but not the phosphodiesterase activity was inhibited reversibly by n-butanol. Sphingomyelin inhibited the microsomal deacylase but not the lysosomal deacylase.

Messenger RNA (poly(A) + RNA) has been prepared from mature skeletal muscle of adult rats by several procedures. These procedures involved oligodT-cellulose chromatography of either muscle polyribosomes (method 1), polysomal RNA isolated by CsCl gradient centrifugation of muscle polysomes (method 2), total muscle RNA isolated by CsCl gradient centrifugation (method 3), or total muscle RNA prepared by phenol extraction (method 4). Each procedure produced RNA in significant yields which sedimented in a broad band (from 4S to greater than 28S) on sodium dodecyl sulfate--sucrose gradients. In addition, poly (A) + RNA from each procedure stimulated protein synthesis in the wheat germ cell-free system. The best combination of yield and messenger activity was obtained for poly (A) + RNA prepared from polysomal RNA by method 2. This poly(A) + RNA preparation stimulated the cell-free synthesis of a number of presumptive myofibrillar proteins, including myosin heavy chain and actin, in the wheat germ system. The presence of the latter protein among the cell-free products was confirmed by DNase I affinity chromatography of appropriate reaction mixtures. Poly(A) + RNA was also isolated from rat cardiac muscle polysomal RNA by method 2. This RNA also directed the synthesis of myofibrillar proteins in the wheat germ system. The relative amounts of the proteins synthesized in the presence of skeletal and cardiac muscle poly(A) + RNA have been compared. The data indicate that the method described is suitable for the isolation of RNA with message activity from mature mammalian muscle.

Plasma membrane vesicles of Saccharomyces cerevisiae were extracted with 1% (w/v) Triton X-100 and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies against the vesicles. Solubilization was shown to be nonselective and 23 immunoprecipitates were observed reproducibly. Four glycoproteins were identified by interaction with concanavalin A and lentil lectin, either immobilized on agarose beads in an intermediate gel or incorporated in the free form in the first dimension gel. One glycoprotein was stainable by the periodic acid--Schiff procedure. None of the glycoproteins had their origin in the cell wall. Five amphiphilic proteins were identified on the basis of charge-shift and hydrophobic interaction crossed immunoelectrophoresis as well as [14C]Triton X-100 and Sudan black B binding. Three of the amphiphilic proteins were also glycoproteins. Based on the carbohydrate content and amphiphilic properties of the proteins, purification schemes using concanavalin A-Sepharose and phenyl-Sepharose were proposed. Trial separations using 1-mL columns were monitored by fused rocket and crossed immunoelectrophoresis.

Sexual development of a homothallic strain of Schizosaccharomyces pombe was monitored by radiolabelling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Of more than 60 bands detected by Coomassie brilliant blue and by autoradiography, about 30 bands synthesized during development were discrete enough for experimental analysis. About a dozen bands are preferentially vegetative, another dozen preferentially developmental. However, vegetative bands as a group are also synthesized during development. Their synthesis is relatively unaffected by low concentrations of cycloheximide or by chloramphenicol and is not temperature sensitive at 37 degrees C nor catabolite repressible. Only band 40 (ca. 40 000 daltons) seems to be exclusively vegetative. The synthesis of developmental bands 13, 18, 24, 30, and alpha, all of which first appear during late-log phase, is catabolite repressible. Developmental band 51 is also synthesized throughout the vegetative phase. The synthesis of bands 24, 30, 51, and alpha is temperature sensitive at 37 degrees C during the development, but that of band 18 is not. The synthesis of band 13 during development is not temperature sensitive, but its earlier synthesis during late-log phase is. The synthesis of all these six developmental bands is immediately inhibited by cycloheximide, but not by chloramphenicol. Their appearance as a group of radioactive bands is greatly diminished in cultures grown in cycloheximide, in chloramphenicol, or in ethidium bromide. Developmental bands 13, 18, 24, and 30 may be called readiness proteins. They first appear prior to the earliest morphological signs of sexual activity. Their developmental synthesis is inhibited by conditions that inhibit sexual development. Such inhibitory conditions include anaerobiosis, restrictive temperature, aging in stationary phase, the presence of inhibitors of cytoplasmic protein synthesis and of mitochondrial function, and catabolite repression. Readiness proteins may be regulating the switch from vegetative metabolism.

The synthesis of CDP-diacylglycerol by CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) has been studied in microsomes isolated from hog mesenteric lymph node lymphocytes. The properties of this enzyme were found to be similar in many respects to that described in rat liver microsomes; however, it is not stimulated by GTP or any other nucleotides under normal assay conditions. The enzyme requires that the phosphatidate be emulsified in a cationic detergent for optimal activity. The quaternary ammonium phospholipids lecithin and sphingomyelin were found to stimulate the formation of CDP-diacylglycerol even in the presence of optimal cationic detergent. Other phospholipids or detergents had no effect or were inhibitory to the reaction. Only in the presence of either lecithin or sphingomyelin did nucleotides such as ATP, GTP, UTP, and ITP stimulate the formation of CDP-diacylglycerol. The Km and Vmax for CTP were found to be 0.6 and 1.2 mM, respectively, while the apparent Km and Vmax for phosphatidate were 0.65 and 1.2 mM. Magnesium was found to be the only metal ion that stimulated the reaction, with an optimal concentration of 20 mM. Fluoride ions at 20 mM inhibited the reaction to the extent of 70%. The enzyme was found to be very unstable when the isolated microsomes were stored at -20 degrees C for 24 h, losing approximately 75% of its activity.

Glycosides of the fully acetylated 3-amino-3,6-dideoxy-L-hexosides in the gluco, galacto, manno, and talo configurations have been treated with chromium trioxide in acetic acid. The alpha-L-methyl glycosides, which exist in the more stable chair conformation 1C4, with an axially oriented aglycon, are resistant to this oxidation, whereas the beta-L-linked glycoside having the gluco configuration (1C4 conformation) and the alpha-L-methyl glycoside having the talo configuration (4C1 conformation), both with equatorially oriented aglycons, are oxidized to the corresponding 5-adulosonates.

The synthesis of polypeptides in Escherichia coli minicells, directed by a pBR322 plasmid and its derivative-carrying bovine growth hormone cDNA insert, was studied. Two polypeptides coded by the ampicillin-resistance (Apr) gene (32 000 and 28 000 daltons) and a tetracycline-resistance (Tcr) polypeptide (36 000 daltons) were identified by insertion inactivation. Two additional polypeptides of 37 000 and 34 000 daltons of as yet unknown function were detected in all extracts regardless of the presence of the Apr or Tcr genes in the plasmid. The pBR322-BGH recombinant plasmid coded for several novel polypeptides, among them one of 46 000 daltons, presumably a fused product of the BGH and beta-lactamase genes. This protein, however, was not secreted into the periplasmic space of the cells as was the beta-lactamase.

The organizations of the genomes of two related species of Asian deer, the Indian (2n = 6 female, 7 male) and Chinese muntjac (2n = 46), were compared at the cytogenetic and molecular levels. These dramatically different karyotypes preserve little apparent G-banding homology. The difference in chromosome number is coincident with a 22% reduction in haploid DNA content from 2.7 to 2.1 pg in the Chinese and Indian muntjac, respectively. The kinetics of reassociation of the Indian muntjac (equivalent Cot = 4285 M-1. s-1) and Chinese muntjac DNA (equivalent Cot - 4362 M-1.s-1) in 2.4 M tetraethylammonium chloride suggests conservation in amount of "single-copy" DNA. Two middle repetitive DNA sequence classes differ in both amount and in degree of repetition between the two species. A middle repetitive frequency component (935-fold repeated) represents 13% of the Indian muntjac DNA. A similar component (644-fold repeated) represents 17% of the Chinese muntjac DNA. Low repetition DNA sequence components (repeated 5- and 50-fold) represent 30 and 40% of the Indian and Chinese muntjac DNAs, respectively. These differences quantitatively account for the 0.6 pg haploid DNA content variation between species. The deletion of middle repetitive DNA has not substantively altered the distribution of restriction endonuclease DNA base composition classes as defined by buoyant density in cesium chloride. These results represent the first time that middle repetitive DNA has been directly implicated in a chromosome rearrangement within the vertebrates.

The incubation of PE2 ((6,7-3H)-labelled 3-propyl ether of estra-1,3,5(10)triene-3, 17 beta-diol (estradiol)) with various subcellular fractions of rat liver indicated that the hepatic metabolism of this compound occurs mainly in the microsomal fraction. In addition to the formation of 3-propyl ethers of estra-1,3,5(10)triene-3-ol-17-one (estrone) and estra-1,3,5(10-triene-3, 16alpha, 17 beta-triol (estriol) directly deriving from PE2, the microsomal proteins carried out the deetherification of the propyl ether group leading to phenolic steroids; among them, estradiol, estrone, and estriol were characterized. Protein-bound and water-soluble metabolites were found; the effects of glutathione and of the incubation conditions were in agreement with the thioconjugation of these derivatives. The microsomal metabolism of PE2, and specially the deetherification reaction, required the presence of oxygen and of NADPH as cofactor, the optimum pH ranging from 7.4 to 8. The participation of cytochrome P450 in these metabolic pathways was shown by a partially inhibited catabolism with carbon monoxide and by a more active metabolism in males than in females and when animals were pretreated with phenobarbital. These results allowed us to conclude that the hepatic deetherification of PE2 is carried out by a microsomal oxidative system which is very similar to the system involved in the demethylation of methyl ethers of estrogens.

Pyridinoline, a cross-linking amino acid of collagen, was degraded by irradiation of ultraviolet light. The decomposition rate varied with pH of the solution and wavelength of irradiation light. The maximum of the degradation rate at individual pH coincides with the ultraviolet absorption maximum. Namely, it was maximally degraded by irradiation at 295 nm in acidic solution and at 325 nm in neutral and alkaline solution. At the optimum wavelength, the photolysis occurred more rapidly in neutral and alkaline solution than in acidic solution. The quantum yield in neutral solution was approximately 0.11 and independent of wavelength. One of the photolysis products was identified as hydroxylysine on an amino acid analyser, indicating that the cleavage of the pyridinium ring occurred.