Plant glucosidase II catalyzes a transglucosylation reaction in addition to the hydrolytic reaction

When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.     

A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate

A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions.     

Arabinoxylan biosynthesis in wheat. Characterization of arabinosyltransferase activity in Golgi membranes

Arabinoxylan arabinosyltransferase (AX-AraT) activity was investigated using microsomes and Golgi vesicles isolated from wheat (Triticum aestivum) seedlings. Incubation of microsomes with UDP-[(14)C]-beta-L-arabinopyranose resulted in incorporation of radioactivity into two different products, although most of the radioactivity was present in xylose (Xyl), indicating a high degree of UDP-arabinose (Ara) epimerization. In isolated Golgi vesicles, the epimerization was negligible, and incubation with UDP-[(14)C]Ara resulted in formation of a product that could be solubilized with proteinase K. In contrast, when Golgi vesicles were incubated with UDP-[(14)C]Ara in the presence of unlabeled UDP-Xyl, the product obtained could be solubilized with xylanase, whereas proteinase K had no effect. Thus, the AX-AraT is dependent on the synthesis of unsubstituted xylan acting as acceptor. Further analysis of the radiolabeled product formed in the presence of unlabeled UDP-Xyl revealed that it had an apparent molecular mass of approximately 500 kD. Furthermore, the total incorporation of [(14)C]Ara was dependent on the time of incubation and the amount of Golgi protein used. AX-AraT activity had a pH optimum at 6, and required the presence of divalent cations, Mn(2+) being the most efficient. In the absence of UDP-Xyl, a single arabinosylated protein with an apparent molecular mass of 40 kD was radiolabeled. The [(14)C]Ara labeling became reversible by adding unlabeled UDP-Xyl to the reaction medium. The possible role of this protein in arabinoxylan biosynthesis is discussed.     

The biosynthesis of ovothiol A (N-methyl-4-mercaptohistidine). Identification of S-(4'-L-histidyl)-L-cysteine sulfoxide as an intermediate and the products of the sulfoxide lyase reaction.

Crude extracts of Crithidia fasciculata catalyse the formation of 4-mercapto-L-histidine, an intermediate in the biosynthesis of ovothiol A (N1-methyl-4-mercaptohistidine), in the presence of histidine, cysteine, Fe2+ and pyridoxal phosphate. This activity was present in a 35-55% ammonium sulfate fraction that was shown to produce a transsulfuration intermediate in the absence of pyridoxal phosphate. The transsulfuration intermediate was isolated and identified as S-(4'-L-histidyl)-L-cysteine sulfoxide. The synthase activity, partially purified by anion-exchange chromatography, was shown to require oxygen and could be used to synthesize a number of isotopically labeled S-(4'-L-histidyl)-L-cysteine sulfoxides. Sulfoxide lyase activity was partially resolved from the synthase by anion-exchange chromatography. The phenylhydrazone of the product derived from the cysteine moiety of the sulfoxide coeluted with the phenylhydrazone of pyruvate on HPLC, but this assignment could not be confirmed by mass spectral analysis. S-(4'-[14C]L-histidyl)-[U-13C3,15N]L-cysteine sulfoxide was synthesized and converted to products of the lyase reaction in the presence of lactate dehydrogenase and NADH. The 13C-labeled product was identified by 13C-NMR spectroscopy as lactate and the primary product of the lyase reaction is therefore pyruvate. With S-(4'[3H]L-histidyl)-[14C]L-cysteine sulfoxide as the substrate [14C]lactate, [14C]cysteine and [3H]4-mercaptohistidine could be detected as products of the lyase reaction, but the sum of the two thiol species exceeded the amount of sulfoxide substrate used. Evidence is presented that this anomaly was due to the utilization of sulfur from dithiothreitol for the formation of cysteine.     

Biosynthesis of the internal thioester bond of the third component of complement

C3-translational product, which was synthesized with rabbit liver mRNA in a reticulocyte lysate protein-synthesizing system, did not react with [14C]methylamine, indicating the lack of an internal thioester bond. Instead, the C3-translational product reacted with iodo[1-14C]acetamide, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate after immunoprecipitation of the product, indicating the presence of a reactive thiol group. When the C3-translational product was treated with rabbit liver homogenate, the product acquired reactivity with [14C]methylamine and lost the reactivity with iodo[1-14C]acetamide. Thus, the liver homogenate seemed to contain a factor (or factors) required for the formation of an internal thioester bond. The factor was partially purified from the liver homogenate by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-cellulose.     

Utilization of nucleoside diphosphate glucoses in developing cotton fibers

The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP-¹⁴C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-¹⁴C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers.     

A new radiochemical assay for argininosuccinase with purified [14C]argininosuccinate

A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with ¹⁴C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-¹⁴C]arginine formed is measured as the sum of radioactivity in [U-¹⁴C]ornithine and [¹⁴C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [¹⁴C]Argininosuccinate was synthesized enzymatically from l-[U-¹⁴C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions.     

The biosynthesis of valine from isobutyrate by Peptostreptococcus elsdenii and Bacteroides ruminicola

1. Growing cultures of Peptostreptococcus elsdenii and Bacteroides ruminicola incorporate (14)C from [1-(14)C]isobutyrate into the valine of cell protein. With P. elsdenii some of the (14)C is also incorporated into leucine. 2. Crude cell-free extracts of both organisms in the presence of glutamine, carbon dioxide and suitable sources of energy and electrons incorporate (14)C from [1-(14)C]isobutyrate into valine but not into leucine. 3. With extracts of P. elsdenii treated with DEAE-cellulose the reaction is dependent on ATP, CoA, thiamin pyrophosphate, molecular hydrogen and a low-potential electron carrier (ferredoxin, flavodoxin or benzyl viologen). 4. The same extracts incorporate (14)C from NaH(14)CO(3) into valine in the presence of isobutyrate plus ATP, CoA, glutamine and ferredoxin; isobutyryl-CoA or isobutyryl phosphate plus CoA will replace the isobutyrate plus CoA and ATP. With acetyl phosphate in place of isobutyryl phosphate, (14)C is incorporated into alanine. With isovalerate or 2-methylbutyrate in place of isobutyrate, (14)C is incorporated into leucine and isoleucine respectively. 5. When carrier 2-oxoisovalerate is added to the carboxylating system (14)C from [1-(14)C]isobutyrate passes into the oxo acid fraction. 6. It is concluded that these two organisms form valine from isobutyrate by the sequence isobutyrate-->isobutyryl-CoA-->2-oxoisovalerate-->valine and that the reductive carboxylation of isobutyrate is catalysed by a system similar to the pyruvate synthetase of clostridia and photosynthetic bacteria.     

Dolichol kinase activity: a key factor in the control of N-glycosylation in inner mitochondrial membranes

Inner mitochondrial membranes from liver contain a dolichol kinase which required CTP as a phosphoryl donor. Kinase activity was linear with protein concentration and unlike other reported kinases, activated almost equally well by Mg2+, Mn2+ or Ca2+. Thin-layer chromatography showed that the reaction product co-migrated with authentic dolichyl monophosphate. The phosphorylation of dolichol did not occur in presence of ATP, GTP or UTP but required exogenous dolichol for maximal activity. Newly synthesized [3H]dolichyl monophosphate has been shown to be glycosylated in the presence of GDP[14C]mannose or UDP[14C]glucose. The double labeled lipids formed by the sugar nucleotide-dependent reactions were identified respectively as [14C]mannosylphosphoryl[3H]dolichol and [14C]glucosylphosphoryl [3H]dolichol. These results are discussed in terms of regulation of N-glycosylation processes in inner mitochondrial membranes from liver.     

Biosynthesis of Glycosyldiglycerides in Mycobacterium smegmatis

A particulate enzyme preparation from Mycobacterium smegmatis catalyzes the transfer of [(14)C]galactose from uridine 5'-diphosphate (UDP)-[(14)C]galactose and of [(14)C]glucose from UDP-[(14)C]glucose into chloroform-soluble products. The radioactive neutral lipids were purified by passage through diethylaminoethyl-cellulose, followed by thin-layer chromatography. When UDP-glucose was used as substrate, two major radioactive lipids were obtained; one had a hexose-glucose-glycerol ratio of 1:1:1. The second product had a hexose-glycerol ratio of 2:1 and, in addition to glucose, contained lesser amounts of mannose and galactose. With UDP-galactose as substrate, two radioactive products were observed that were chromatographically indistinguishable from the [(14)C]glucosyl-labeled mono- and diglycosyldiglyceride. Palmitate and oleate were the predominant fatty acid constituents in these lipids and were present in equimolar amounts in all of the products examined. The products have thus been identified as monoglycosyldiglyceride and a diglycosyldiglyceride containing glucose as the major hexose along with mannose and galactose. Properties of the galactosyl and glucosyl transferases are described.     

Measurement of the metabolism of energy substrates in individual bovine blastocysts

Individual blastocysts from cows were cultured for 3 h under 5% CO2 in air, in 4 microliters droplets of Ham's F-10 medium containing D-[5-3H]glucose, D-[1-14C]-glucose, D-[6-14C]glucose, [2-14C]pyruvate, or L-[U-14C]glutamine, and with or without 2,4-dinitrophenol (DNP) or phenazine ethosulphate (PES). The 14CO2 or 3H2O produced were collected by exchange with an outer bath of 400 microliter 25 mM-NaHCO3. All combinations of substrate and treatment (control, DNP or PES) produced measurable quantities of labelled product except for D-[6-14C]glucose in the presence of PES. Untreated and DNP-treated embryos developed normally during a subsequent 48-h culture period in fresh medium, but PES-treated embryos degenerated. Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate. Conversely, DNP has no significant effect on glucose metabolism, indicating that glycolysis is blocked in the bovine blastocyst due to a lack or inhibition of pyruvate kinase. The production of 14CO2 from D-[1-14C]glucose increased significantly in the presence of PES, indicating that the activity of the pentose shunt is less than maximal.     

Purification and characterization of OleA from Xanthomonas campestris and demonstration of a non-decarboxylative Claisen condensation reaction

OleA catalyzes the condensation of fatty acyl groups in the first step of bacterial long-chain olefin biosynthesis, but the mechanism of the condensation reaction is controversial. In this study, OleA from Xanthomonas campestris was expressed in Escherichia coli and purified to homogeneity. The purified protein was shown to be active with fatty acyl-CoA substrates that ranged from C(8) to C(16) in length. With limiting myristoyl-CoA (C(14)), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [(14)C]myristoyl-CoA, the other products were identified as myristic acid, 2-myristoylmyristic acid, and 14-heptacosanone. 2-Myristoylmyristic acid was indicated to be the physiologically relevant product of OleA in several ways. First, 2-myristoylmyristic acid was the major condensed product in short incubations, but over time, it decreased with the concomitant increase of 14-heptacosanone. Second, synthetic 2-myristoylmyristic acid showed similar decarboxylation kinetics in the absence of OleA. Third, 2-myristoylmyristic acid was shown to be reactive with purified OleC and OleD to generate the olefin 14-heptacosene, a product seen in previous in vivo studies. The decarboxylation product, 14-heptacosanone, did not react with OleC and OleD to produce any demonstrable product. Substantial hydrolysis of fatty acyl-CoA substrates to the corresponding fatty acids was observed, but it is currently unclear if this occurs in vivo. In total, these data are consistent with OleA catalyzing a non-decarboxylative Claisen condensation reaction in the first step of the olefin biosynthetic pathway previously found to be present in at least 70 different bacterial strains.     

NAD(P)H-dependent 6'-deoxychalcone synthase activity in Glycyrrhiza echinata cells induced by yeast extract

The crude extract prepared from Glycyrrhiza echinata cells treated with yeast extract catalyzed the formation of liquiritigenin (5-deoxyflavanone) and isoliquiritigenin (6'-deoxychalcone) in addition to naringenin (5-hydroxyflavanone) when incubated with 4-coumaroyl-CoA and malonyl-CoA in the presence of high concentrations (0.1 mM or higher) of NADPH. Incubation without NADPH, or with low concentrations (0.01 mM or lower), gave only naringenin as a reaction product. With NADH (1 mM), the major product was naringenin accompanied by a small quantity of liquiritigenin. The initial product of the assay with 1 mM NADPH was isoliquiritigenin, indicating a reaction catalyzed by 6'-deoxychalcone synthase (DOCS). Subsequent formation of liquiritigenin was attributed to the presence of chalcone isomerase in the crude extract. The results constitute the first demonstration in vitro of DOCS activity which, in G. echinata cells and other leguminous plants, is involved in the biosynthesis of retrochalcone and 5-deoxyisoflavonoid-derived phytoalexins.     

Pyruvate metabolism by Anaplasma marginale in cell-free culture.

Partially purified Anaplasma marginale initial bodies were cultivated in a cell-free system in the presence of [3-14C]pyruvate for 24 or 48 h. Experiments showed that a significant portion of the pyruvate supplied to the cultures was incorporated into initial body components. Label incorporation was reduced by 72% in the presence of oxytetracycline. Fractionation and chromatography of the organisms revealed radioactive incorporation as alanine. This is the first report of de novo amino acid synthesis by A. marginale demonstrating that the rickettsia is capable of using pyruvate, an erythrocyte glycolytic product, in its metabolism.     

Rapid radiometric method for detection of Salmonella in foods.

A radiometric method for the detection of Salmonella in foods has been developed which is based on Salmonella poly H agglutinating serum preventing Salmonella from producing 14CO2 from [14C]dulcitol. The method will detect the presence or absence of Salmonella in a product within 30 h compared to 4 to 5 days by routine culture methods. The method has been evaluated against a routine culture method using 58 samples of food. The overall agreement was 91%. Five samples negative for Salmonella by the routine method were positive by the radiometric method. These may have been false positives. However, the routine method may have failed to detect Salmonella due to the presence of large numbers of lactose-fermenting bacteria which hindered isolation of Salmonella colonies on the selective agar plates.     

14C]acrylonitrile: preparation via a stable tosylate intermediate and quantitative reaction with amine residues in collagen

A simple, convenient synthetic procedure for [14C]acrylonitrile is described. Na14CN is used as the radioactive starting material. Small (milligram) amounts are converted to 3-[14C]Hydroxypropionitrile by a substitution reaction with 2-chloroethanol. 3-[14C]Hydroxypropionitrile is then tosylated, and the specific activity of this intermediate product is easily determined using its uv extinction coefficient and scintillation counting. [14C]Acrylonitrile is obtained rapidly on distillation by heating the tosylate in the presence of a high boiling tertiary amine base catalyst. The tosylate intermediate can be stored, in contrast to radioactive acrylonitrile, which is unstable. The reaction of acrylonitrile with lysine, hydroxylysine, and histidine residues in human Achilles tendon collagen, as well as chromatographic separation and identification of the carboxyethyl derivatives of these amino acids, is also described.     

Metabolism of S-adenosylhomocysteine and S-tubercidinylhomocysteine in neuroblastoma cells.

The metabolism of the methylase product inhibitor S-adenosylhomocysteine and its 7-deaza analogue S-tubercidinylhomocysteine has been studied in cultured N-18 neuroblastoma cells. The latter compound, designed to resist metabolic degradation, has been shown to be inert under the same conditions where S-adenosylhomocysteine is rapidly and extensively degraded. The product analyses elucidated by high-performance liquid chromatography indicate that the primary route of S-[8-(14)C]adenosylhomocysteine metabolism in these cells leads to adenosine. This product does not accumulate but is rapidly converted to nucleotides or oxypurines by the action of adenosine kinase and adenosine deaminase, respectively. The presence of the potent adenosine deaminase inhibitor coformycin leads to a pronounced inhibition of oxypurine formation, an increase in nucleotide formation, and a slight accumulation of the primary metabolic products adenosine and adenine.     

Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis.

A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.     

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.     

Pharmacokinetics of C14-olivomycin in the body of mice with lymphosarcoma (L10-1 strain)]

The pharmacokinetics of C14-olivomycin after its single intravenous administration to mice with lymphosarcome (LIO-I) was studied. It was shown that according to the specific radioactivity the organs may be placed in the following order: I hour after the antibiotic administration-the blood, liver, spleen, thymus, tumor, muscle; 3 hour after the administration-the liver, spleen, thymus, blood, tumor, muscle. Accumulation of olivomycin in the mouse organs was mainly in direct dependence on the dose of the antibiotic administered. Chromatography of the substances extraceted with ethylacetate from the urine collected at various periods after C14-olivomycin administration showed the presence of a new radioactive product (Rf 0.35-0.37) in addition to the unchanged antibiotic (Rf 0.53). Bioautographic analysis of the chromatograms showed that the product of C14-olivomycin conversion preserved its biological activity. The analysis of the substances extracted with ethylacetate from the liver, spleen and tumors 3 hours after the antibiotic administration reveiled (except of the liver) the presence of a spot with Rf corresponding to that of the initial drug.