Allozyme variation in the stingless bee Trigona fuscobalteata (hymenoptera,apidae) from Peninsular Malaysia

• 1.1. A population of Trigona fuscobalteata from Peninsular Malaysia was analysed for genetic variation at 9 gene-enzyme systems comprising 13 loci.
• 2.2. Two gene-enzyme systems (phosphoglucomutase and isocitrate dehydrogenase) were polymorphic in the 20 colonies studied.
• 3.3. Isocitrate dehydrogenase was represented by duplicate genes.
• 4.4. The number of loci for several enzyme systems appeared to be different from that reported for the Australian stingless bees.

     

Purification and characterization of the d-lactate dehydrogenase from Leuconostoc lactis

• 1.1. The d-lactate dehydrogenase from Leuconostoc lactis has been purified in high yield.
• 2.2.The enzyme is a dimer of subunits of M_r = 39,000 and each subunit contains a single thiol group. The N-terminal residue is methionine.
• 3.3. The amino acid composition has been determined and is typical of that of a soluble globular protein.

     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

NADPH/NADP ratio could regulate the glyoxylate cycle in Tetrahymena pyriformis

• 1.1. The glyoxylic acid cycle pathway could be regulated through the modulation of the isocitrate dehydrogenase-NADP activity. This enzyme is inhibited by NADPH.
• 2.2. The effect on the glyoxylate cycle flux of variations in the rate of the NADPH-consuming pathways has been studied.
• 3.3. Increase in the rate of NADPH-consuming activity by addition of H₂O₂ produces inhibition of the glyoxylate cycle and decrease in the NADPH/NADP ratio.
• 4.4. These results suggest that the glyoxylate flux in Tetrahymena could be modulated by regulation of NADP-dependent isocitrate dehydrogenase by the NADPH/NADP ratio.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

Allozyme variation in the solanaceous fruit fly Bactrocera latifrons (insecta: diptera: tephritidae) from peninsular malaysia

• 1.1. Bactrocera latifrons fruit flies recovered from four solanaceous fruits (Capsicum annuum, Lycopersicon esculentum, Solanum pseudocapsicum and Solanum melongena) in Peninsular Malaysia were analyzed for a total of 15 gene-enzyme systems comprising 21 loci.
• 2.2. Eleven loci—aAdh, Aldox, Ald, Est-F, Est-S, Hk-F, Ldh, cMdh, Me, Pep-A and Pep-C—were invariant.
• 3.3. Of the polymorphic loci, cathodal alcohol dehydrogenase, glucose phosphate isomerase, glycerol-3-phosphate dehydrogenase, hydroxybutyrate dehydrogenase, isocitrate dehydrogenase, anodal malate dehydrogenase and phosphoglucomutase were represented by two alleles each, while hexokinase-S, peptidase-B and phosphogluconate dehydrogenase were represented by three alleles each.
• 4.4. The proportion of polymorphic loci ranged from 0.28 to 0.33, while the mean heterozygosity ranged from 0.04 to 0.13.
• 5.5. The genetic variability is associated with the host range.

     

Purification and partial characterization of an AMP deaminase from the marine invertebrate Palaemon serratus

• 1.1. AMP deaminase from Palaemon serratus tail muscle was partially purified by chromatography on cellulose phosphate.
• 2.2. Muscle homogenates expressed very low enzyme activities and the presence of ATP was necessary to detect AMP deaminase. The specific activity and substrate affinity of the purified enzyme were also very low.
• 3.3. The purified prawn muscle AMP deaminase was contaminated by contractile proteins, one of the major contaminants being actin.
• 4.4. The enzyme displayed a very high affinity for actomyosin which was only partially abolished by pyrophosphate.

     

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
• 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
• 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
• 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower M_r, Stokes-radius and diffusion coefficient.
• 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the M_r of the subunits were unaffected.
• 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
• 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
• 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.

     

Some properties of the nadp+dependent isocitrate dehydrogenase of rat and pig ovary

• 1.1. Cytosolic isocitrate dehydrogenase from rat and pig ovaries differs from that described in other tissues. It is more labile, has higher K_m-values for substrates, higher optimal pH, and its activity is modulated by varying ratios of Ca²⁺: Mg²⁺. The ovarian enzyme exhibits acute and long term responses to in vivo administration of gonadotropins.
• 2.2. A partial purification yielding a 70-fold increase in specific activity of this NADP⁺ dependent enzyme is described. The enzyme mol wt = 58,000 ± 3000 daltons and pI = 4.9.
• 3.3. The unique properties of the enzyme are discussed in relation to its probable metabolic role in ovary.

     

Isolation and partial amino acid sequence of A 78 kDa porcine gastrin-binding protein

• 1.1. A 78 kDa protein (p78) has been partially purified from washed membranes isolated from the corpus of porcine gastric mucosa. The purification was monitored by covalent cross-linking of iodinated [Nle¹⁵]-gastrin; 17.
• 2.2. A single N-terminal sequence extending for 33 amino acids was obtained from the p78 preparation. Partial sequences totalling 192 amino acids were also obtained from 14 tryptic and 3 Staphylococcal V8 peptides.
• 3.3. 10 peptides plus the N-terminal sequence were derived from a previously unsequenced protein which was distantly related to the product of the E. coli fadB gene (Baldwin G. S. (1993) Comp. Biochem. Physiol. 104B, 55–61). The remaining 7 peptides were derived from the gb-subunit of the gastric H⁺/K⁺-ATPase.
• 4.4. The gastrin-binding activity remained in association with p78, and could be separated from the P-subunit of the gastric H⁺K⁺-ATPase, during chromatography on tomato lectin-Sepharose.
• 5.5. We conclude that p78 binds gastrin, and is a novel member of the enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase family of enzymes.

     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Purification,characterization and kinetic mechanism of glucose-6-phosphate dehydrogenase from mouse liver

• 1.1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE.
• 2.2. The native and subunit molecular weight were 117 and 31 kDa respectively.
• 3.3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism.
• 4.4. The reduced K_m values for the substrates favour the operativity of the enzyme. The “fine control” of the enzymatic activity was exerted by the NADPH, whose K_i is several fold lower than the in vivo concentration.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Human spleen cathepsin D: Its characterization and localization in human spleen

• 1.1. The cathepsin D was purified 1830-fold under mild conditions by a rapid procedure, based on two-step affinity chromatography.
• 2.2. Its molecular weight, amino acid composition and substrate specificity were shown to display minor differences from materials of other origins.
• 3.3. Inhibition with thiol compounds was found to be a specific phenomenon of the cathepsin D from the human spleen.
• 4.4. Production of antiserum specific for purified cathepsin D was demonstrated by immunodiffusion test, an immunoadsorbent column and immunoblotting of the crude enzyme in SDS gel.
• 5.5. In an immunocytochemical study, the antigenic sites for this enzyme were found to be localized in the reticuloendothelial system of the human spleen.
• 6.6. The role of this enzyme in human spleen cell was discussed.

     

Purification and properties of glycollate oxidase from Lemna minor L.

• 1.1. Glycollate oxidase has been purified to apparent homogeneity from Lemna minor L. grown on medium containing 7mM NO⁻₃.
• 2.2. The enzyme is a highly basic protein with a sub-unit molecular weight of 42,000 and a holoprotein molecular weight of 250,000.
• 3.3. The Lemna enzyme is a flavoprotein with a broad specificity for straight chain α-hydroxy acids, the preferred substrate being glycollate.
• 4.4. It is also competitively inhibited by oxalate and phenyllactate.
• 5.5. A comparison is drawn between the physical properties of glycollate oxidase from a number of higher plants and the degree of sub-unit aggregation in the resulting protomers.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.