Purification and characterization of a manganese containing superoxide dismutase from bovine heart mitochondria

• 1.1. A manganese containing superoxide dismutase from bovine heart mitochondria was isolated and characterized.
• 2.2. It has a molecular weight of about 86,000 and is composed of 4 noncovalently bound subunits of equal size.
• 3.It appears to contain 2 mole manganese per mole enzyme.
• 4.The carbohydrate content is very low.
• 5.The specific activity and amino acid composition are similar to those of other mitoehondrial superoxide dismutases.
• 6.The enzyme forms complexes with ampholytes and can therefore not be analysed by isoelectric focusing.

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

Soluble and immobilized RP. palustris rhodanese—II. Some particular kinetic behaviour

• 1.1. Kinetic studies were carried out on the soluble and immobilized Rhodanese.
• 2.2. The soluble enzyme showed a typical Michaelis-Menten behaviour, an inhibitory effect was observed at high thiosulphate and cyanide concentrations.
• 3.3. The product sulphite was also an inhibitor, instead thiocyanate increased the enzyme velocity when it was added to the incubation mixture.
• 4.4. A ping-pong mechanism was proposed for Rp. palustris Rhodanese with a stable (free enzyme: E) and an unstable (sulfur substituted enzyme: ES) kinetic enzyme form.
• 5.5. The insolubilized Rhodanese presented an unusual kinetic behaviour, with sigmoid shape substrate profiles and non-linear double reciprocal plots.
• 6.6. From the empirical Hill equation, positive cooperativity (n>1) was found for both substrates.

     

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.

     

Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

Fatty acid synthetase from pigeon red blood cells

• 1.1. Fatty acid synthetase has been purified 200-fold from pigeon erythrocytes.
• 2.2. The enzyme gave 2 major staining bands on disc gel electrophoresis corresponding to the complex and dissociated forms of the enzyme.
• 3.3. Sucrose density gradient centrifugation of the enzyme showed only one sedimenting peak and high performance liquid chromatography also showed only 1 major light absorbing peak.
• 4.4. The molecular weight of the enzyme was estimated to be 300,000–330,000 and the enzyme is comprised of 2 subunits of similar molecular weights.
• 5.5. The red blood cell fatty acid synthetase was found to be immunochemically nonidentical with the liver fatty acid synthetase.

     

Involvement of lysine residue in the nucleotide binding of pigeon liver malic enzyme: modification with affinity label periodate-oxidized nadp

• 1.1. Periodate-oxidized NADP, a competitive inhibitor of malic enzyme with respect to NADP. inactivate the enzyme in mild conditions.
• 2.2. The inactivation is due to the modification of an essential lysine residue.
• 3.3. Two molecules of reagent were found to be incorporated into the enzyme tetramer after extensive modification.
• 4.4. Complete protection of malic enzyme from the oxidized NADP inactivation was afforded by NADP and its analogues.
• 5.5. The modified enzyme showed increased apparent Michaelis constant for the nucleotide coenzymes but the maximum velocity was decreased.
• 6.6. The binding between the modified enzyme and NADPH was impaired.

     

Studies on tryptophan pyrrolase of Schistocerca gregaria försk. (Orthoptera)

• 1.The trytophan pyrrolase activity of central fat bodies of S. gregoria hoppera was studied.
• 2.The enzyme system appears to be similar to that of mammalian liver.
• 3.The enzyme was localized only in central fat bodies.
• 4.Extracts of other body parts can mimic an enzyme activity because of a degradation of ommochromes in the enzyme test.

     

Kinetic studies of the transphosphorylation reactions catalyzed by alkaline phosphatase from E. coli: Hydrolysis of p-nitrophenyl phosphate and o-carboxyphenyl phosphate in presence of tris

• 1.1. Transphosphorylation of p-nitrophenyl phosphate and o-carboxyphenyl phosphate to Tris, has been studied at alkaline and acid pH.
• 2.2. The rate of release for all reactions products was Tris-dependent for both substrates, with a slight maximum for phenol at alkaline pH. These dependences have been analyzed from a mechanistic standpoint.
• 3.3. Individual constants of rate of a simple transphosphorylation mechanism have been determined.
• 4.4. At high Tris concentrations (> 1.0 M) a slight competitive inhibition has been observed.
• 5.5. Inhibition in NH₄⁺-NH₃Cl buffer has been found at alkaline pH but not at acid pH. It would therefore seem that the non-protonated NH₂ group of Tris is responsible for inhibition.
• 6.6. The results suggest the formation of complexes between Tris and the enzyme. Other possible alternatives are also analyzed.

     

Purification and characterization of the endonuclease present in Physalia physalis venom

• 1.1. Physalia physalis nematocyst venom contains a DNase which has a non-specific endonucleolytic action.
• 2.2. This enzyme has an approximate molecular weight of 75,000 daltons.
• 3.3. The enzyme can cleave DNA over a wide pH range with an optimum near neutrality.
• 4.4. The enzyme is thermolabile and its activity can be stimulated by 80 nM NaCl or 10 mM MgCl₂.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Reductive metabolism of D-glyceraldehyde,L-glyceraldehyde and dihydroxyacetone in rat liver: Identification of alcohol dehydrogenase as the main responsible enzyme

• 1.1. The paper describes NADH- and NADPH-dependent enzyme activities in rat liver which catalyse the reduction of the following substrates: d-glyceraldehyde, l-glyceraldehyde and dihydroxyacetone. Test conditions for the optimal rates of the oxidoreductase reactions are described.
• 2.2. As a test of metabolic relevance of these activities the hormonal status of the rats was changed by pretreatment with alloxan.
• 3.3. This lowers all described activities if the concentration of blood glucose is increased. But there is also a range of elevated activities which are not associated with changes in the glucose concentration.
• 4.4. It is shown that rat liver alcohol dehydrogenase (EC 1.1.1.1) 2 is the enzyme which catalyses the reduction of the substrates named above and also of acetaldehyde with NADH and NADPH. The preparation and characterization of the enzyme are described.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

A comparative study of porphyrin synthesis by whole blood and haemolysates from different mammalian species

• 1.1. There is no uniform pattern of porphyrin synthesis by whole blood and haemolysates, between the different mammalian species studied.
• 2.2. Coproporphyrin, is the dominant porphyrin synthesised by intact red cells.
• 3.3. Uroporphyrin synthesis increases significantly in the majority of species when the cells are haemolysed.
• 4.4. In the mouse large amounts of protoporphyrin are synthesised by intact red cells which increases further on haemolysis.
• 5.5. The low porphyrin synthesising capacity of cow and sheep red cells is not due to any rate-limiting activity of the enzyme ALA-dehydratase.
• 6.6. The dog has a pattern of porphyrin synthesis and excretion similar to that found in the rabbit, and the possibility exists, that a similar energy-dependent carrier mechanism for movement of uroporphyrin and coproporphyrin across red cell membranes found in the rabbit may be present in this species.
• 7.7. The findings may be of significance in the interpretation of porphyrin excretion patterns in experimental porphyria.

     

Purification and partial characterization of the alkaline phosphatase from Allolobophora caliginosa

• 1.1. A purification of the enzyme from the starting material was achieved by means of butanol and acetone fractionations and, successively, by DEAE cellulose and Sephadex G-200 chromatographies.
• 2.2. Two enzymatic forms were separated; they showed various similar characteristics but differed greatly in specific activity.
• 3.3. It is probable that in A. caliginosa a sole alkaline phosphatase form exists and the less active fraction is partly denatured enzyme.
• 4.4. It is not completely possible to exclude the existence of two isoenzymes.

     

Kinetic properties of cat liver microsomal glucose-6-phosphatase

• 1.1. Cat liver microsomes contain the multifunctional enzyme glucose-6-phosphatase.
• 2.2. High specificity was shown for the phosphohydrolase as well as for the transferase activity.
• 3.3. Both activities have high V_max values determined in optimized conditions.
• 4.4. The phosphate transfer with carbamyl-phosphate as a phosphoryl donor and d-glucose as acceptor is consistent with a random mechanism in which the binding of one substrate decreases the enzyme's affinity for the second substrate.

     

Regulatory properties of rabbit liver phosphorylase kinase

• 1.1. Purified native rabbit liver phosphorylase kinase becomes activated during the assay of its activity while low molecular weight forms of the same enzyme do not.
• 2.2. The activation requires ATP and maganesium ions, suggesting the phosphorylation of the enzyme by a protein kinase as the mechanism involved.
• 3.3. The activation of the enzyme can be reverted by the action of a type 1 protein phosphatase isolated from the same tissue.
• 4.4. The activation can also be catalyzed by the catalytic subunit of cAMP-dependent protein kinase in a process that requires a much lower ATP concentration to proceed.
• 5.5. The activation is believed to be due to an autocatalytic phosphorylation of phosphorylase kinase itself. In support of this hypothesis are the regulation of the process through calcium ions, the low levels of endogenous protein kinase detected in the purified preparation, the high ATP concentrations required in the absence of cAMP dependent protein kinase and the fact that the process cannot be blocked by an excess of the heat stable inhibitor specific for the later enzyme.
• 6.6. The low molecular weight forms of the enzyme on their side are not affected by the action of neither protein phosphatase 1 nor cyclic AMP dependent protein kinase.
• 7.7. Both activated and nonactivated phosphorylase kinase are partially dependent on calcium ions, the affinity of the former being higher than that of the latter. The low molecular forms do not require calcium ions to express their activity.

     

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.

     

Phospholipase A activity of culture supernatants from Streptococcus mutans strain 6715

• 1.1. Phospholipase A activity was found in the culture broth of growing cultures of Streptococcus mutans strain 6715.
• 2.2. The amount of enzyme activity was proportional to the cell density of the cultures.
• 3.3. The enzyme had a pH optimum of 7.0 and was inactivated at temperatures greater than 45°C.
• 4.4. The enzyme was Ca²⁺-dependent, since both EDTA and EGTA were inhibitory and Ca²⁺ was stimulatory.
• 5.5. Analysis of the fatty acid products resulting from the enzyme's action on 1-palmitoyl-2-oleoyl phosphatidylcholine indicated the enzyme to be a phospholipase A₁, (EC 3.1.1.32).

     

Purification and some particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.