Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared repressed while sulphite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) were derepressed and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3-phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase.Abbreviations APS adenylylphosphosulphate - MVH reduced methylviologen - OAS O-acetyl-l-serine - PAPS 3-phospho adenylylphosulphate     

Recognition of sodium- and potassium-dependent adenosine triphosphatase on mouse lymphoid cells by means of a monoclonal antibody

Summary Previous evidence has established the similarity between (Na⁺+K⁺)-ATPase (ATP phosphohydrolase, EC.3.6.1.3) and the antigen recognized by the rat antimouse monoclonal antibody anti-BSP-3. This antibody has been used for investigation of the surface expression and biochemical analysis of the enzyme in different mouse lymphoid populations. The BSP-3 determinant is found on almost all thymocytes and concanavalin A-induced thymocytes, to a lesser extent on bone marrow cells and also on a minor population of spleen cells. Spleen cells from athymic mice are negative. The (Na⁺+ K⁺)-ATPase purified from mouse thymus by affinity chromatography migrates in SDS-polyacrylamide gels in the form of two polypeptide chains of 105000 and 51000 daltons. Chains of the same molecular weight, fractionated on SDS-PAGE from microsomes of mouse thymuses, are shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Immunoprecipitation with anti-BSP-3 from surface iodinated thymocytes yields only the small subunit. Comparison of the chains isolated from thymus and brain shows molecular weight differences in both subunits. These results, and variations in the reactivity pattern of the anti-BSP-3 antibody on several cell types, may indicate a possible heterogeneity of the (Na⁺+K⁺)ATPase expressed by various tissues and cells.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Effect of carbon source on enzymes involved in glycerol metabolism inNeurospora crassa

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Enzyme activities in concentrated solutions of glycinebetaine and other solutes

The activities of a number of enzymes in concentrated solutions of glycinebetaine and other solutes have been studied. Glycinebetaine, in contrast to electrolytes such as NaCl, was found to be noninhibitory up to 500 mM. This is compatible with the postulated role of glycinebetaine in cytoplasmic osmoregulation. Partial protection against NaCl inhibition was afforded by glycinebetaine in some cases. More detailed studies on glycinebetaine —NaCl-enzyme interactions were carried out using malate dehydrogenase (decarboxylating) from Hordeum vulgare.Abbreviations TES N-tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid - MES 2[N-Morpholino]ethane sulphonic acid     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

On the specificity of the uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: Diamino acid ligase of Bifidobacterium globosum

The peptidoglycan of Bifidobacterium globosum contains ornithine and lysine alternately in the same position of the peptide subunit. The uridine diphospho-N-acetylmuramyl-alanyl-D-glutamic acid: diamino acid ligase of this organism was purified 700-fold. Since the activities for the incorporation of ornithine and lysine into uridine diphospho-N-acetylmuramyl-tripeptide did not separate during purification and since the incorporation of ornithine is competitively inhibited by lysine and vice versa, both ornithine and lysine are assumed to be incorporated by one single enzyme. Studies on the specificity of the ligase toward analogs of ornithine have shown that the enzyme requires a diamino, monocarboxylic acid with 4–6 carbon atoms. Methylation of the -amino group or hydroxylation of the -carbon atom of lysine decreases the competitive properties of the analog, whereas the substitution of the -methylen group by sulfur (S-2-aminoethyl cysteine) results in a highly competitive compound.Abbreviations BSA bovine serum albumine - MurNAc N-acetyl-muramyl - DA diamino acid - Ala-DGlu--L-DA-DAla-D-Ala pentapeptide - Ala-DGlu--LDA tripeptide - Ala-DGlu dipeptide - DSM Deutsche Sammlung von Mikroorganismen - CEM clostridial enrichment medium     

Glyoxysomal and mitochondrial malate dehydrogenase of watermelon (Citrullus vulgaris) cotyledons

Molecular properties of the glyoxysomal and mitochondrial isoenzyme of malate dehydrogenase (EC 1.1.1.37; L-malate: NAD⁺ oxidoreductase) from watermelon cotyledons (Citrullus vulgaris Schrad.) were investigated, using completely purified enzyme preparations. The apparent molecular weights of the glyoxysomal and mitochondrial isoenzymes were found to be 67,000 and 74,000 respectively. Aggregation at high enzyme concentrations was observed with the glyoxysomal but not with the mitochondrial isoenzyme. Using sodium dodecyl sulfate electrophoresis each isoenzyme was found to be composed of two polypeptide chains of identical size (33,500 and 37,000, respectively). The isoenzymes differed in their isoelectric points (gMDH: 8,92, mMDH: 5.39), rate of heat inactivation (gMDH: _1/2 at 40°C=3.0 min; mMDH: stable at 40°C; _1/2 at 60°C=4.5 min), adsorption to dextran gels at low ionic strenght, stability against alkaline conditions and their pH optima for oxaloacetate reduction (gMDH: pH 6.6, mMDH: pH 7.5). Very similar pH optima, however, were observed for L-malate oxidation (pH 9.3–9.5). The results indicate that the glyoxysomal and mitochondrial MDH of watermelon cotyledons are distinct proteins of different structural composition.Abbreviations EDTA ethylene diamine tetraacetic acid - gMDH and mMDH glyoxysomal and mitochondrial malate dehydrogenase, respectively