Purification of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

Purification and properties of hydroxypyruvate reductase from Lemna minor L

• 1.1. Hydroxypyruvate reductase has been purified 193-fold from Lemna minor L. by affinity chromatography on Blue Sepharose.
• 2.2. The enzyme has activity over a broad pH range (optimum pH 6), a K_m hydroxypyruvate of 59 μ M and K_m NADH of 12μM.
• 3.3. Crude extracts of Lemna exhibit substrate inhibition of activity above 1 mM hydroxypyruvate, a property which is lost on purification.
• 4.4. Oxaloacetate inhibits purified preparations of the enzyme and a possible role for such regulation in vivo is discussed.

     

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

• 1.1. Detergent solubilisation of particulate rat liver low K_m cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment.
• 2.2. The detergent solubilised enzyme could be partially purified by anion exchange chromatography.
• 3.3. It displayed a marked tendency to precipitate from solution when detergent was removed.

     

The effect of γ-radiation on the NADP-malic enzyme from mango fruit,Mangifera indica — II. Kinetic and regulatory properties

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in aqueous solution and the effect of γ-irradiation on the catalytic and regulatory properties of the enzyme was investigated.
• 2.2. Significant differences in some of the allosteric properties of the enzyme were found as reflected in the various Hill-coefficients.
• 3.3. Changes in both the kinetic parameters V_max and K_m were observed; suggesting that irradiation leads not only to destruction of the active sites but also to a general denaturation of the enzyme.
• 4.4. The physiological significance of the radiation induced alterations are discussed against the background of ripening and sensescence.

     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Purification,isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes

• 1.1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP)3 isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL.-6B chromatography and isoelectric focusing using carrier ampholytes. pH 4–6.
• 2.2. The isoenzyme has an isoelectric point of 5.00 ± 0.05 and could be purified 33.000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme.
• 3.3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges.
• 4.4. At 4°C the isoenzyme is more stable in the pH range of 7–9 than at acid pH values.
• 5.5. Incubation at 30 and 40°C for 4 hr does not affect the activity of the isoenzyme.
• 6.6. It has a K_m-value of 0.28 mM for phosphoglycolate (PG) as substrate.

     

Lipoxygenase of the thermophilic bacteria thermoactinomyces vulgaris—properties and study on the active site

• 1.1. A lipoxygenase activity was purified from Thermoactinomyces vulgaris and some of its properties were characterized.
• 2.2. The enzyme showed a temperature activity range of 40–55°C with still significant activity over 60°C.
• 3.3. The pH of activity on linoleic acid had a broad range with an optimum at pH 6.0 and a weaker one at pH 11.0.
• 4.4. On arachidonic acid the pattern was narrow bell-shaped with an optimum at pH 6.5.
• 5.5. The purified lipoxygenase from Th. vulgaris showed an apparent K_m of 1 mM and V_max of 0.84 μmol diene/min/mg protein.
• 6.6. It was inhibited by the oxidation products, 9-HPOD and 13-HPOD.
• 7.7. A 160,000 Da molecular weight of the enzyme was determined by molecular filtration. Methionine, tyrosine, tryptophan and cysteine are apparently involved in its activity.

     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Effects of adenine nucleotides on low Km 5′ nucleotidase from human seminal plasma

• 1.1. Low K_m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates.
• 2.2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V_max/K_m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides.
• 3.3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

     

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

• 1.1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine.
• 2.2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5.
• 3.3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site.
• 4.4. The K_I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane.
• 5.5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

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.

     

Isolation and characterization of lipoamide dehydrogenase from mackerel dark muscle

• 1.1. Lipoamide dehydrogenase was purified 1500-fold from mackerel dark muscle.
• 2.2. The enzyme was homogeneous as judged by acrylamide gel electrophoresis in the presence and absence of SDS.
• 3.3. Molecular weights of 102,000 and 55,000 were estimated for the native and denatured enzyme, respectively.
• 4.4. Optimal activity for the enzyme was obtained at around pH 5.7 and enhanced with citri acid.
• 5.5. Loss of activity was less than 5% by incubating the enzyme at 70°C for 20 min.
• 6.6. An apparent K_m of 3.1 × 10⁻³ M was obtained for dl-lipoic acid and 1.5 × 10⁻⁵ M for NADH.
• 7.7. The properties of lipoamide dehydrogenase from mackerel dark muscle observed in this investigation were very similar to those reported for the enzyme from other sources.

     

Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos

• 1.1. Arylsulfatase was extracted from sea urchin (Hemicentrotus pulcherrimus) plutei and purified to electrophoretical homogeneity by means of DEAE-cellulose, acetone fractionation and Sepharose CL-6B, successively.
• 2.2. The molecular weight of this enzyme was approx, 670,000. The molecular weight of a single subunit was approx. 63,000. The K_m value for p-nitrophenyl sulfate was 0.59 mM.
• 3.3. This enzyme was competitively inhibited by the sulfate ion and was classified as the type II arylsulfatase. The pH optimum was between 5.0 and 6.0.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.