Studies on the active centre(s) of rat liver porphyrinogen carboxy-lyase. in vivo effect of hexachlorobenzene on decarboxylation site(s) of porphyrinogens

• 1.1. The role of histidine on the decarboxylation of porphyrinogens of 7-, 6-, and 5-COOH III brought about by porphyrinogen carboxy-lyase (PCL) was studied.
• 2.2. For this purpose hepatic PCL from normal and hexachlorobenzene (HCB) treated rats were modified with diethylpyrocarbonate.
• 3.3. The results indicated that the enzyme from both normal and porphyric animals had histidine at the binding sites of all the porphyrinogens assayed.
• 4.4. Comparative studies between the enzyme from normal and porphyric rats suggested that in vivo HCB treatment affected the active site for the decarboxylation of 7-, 6- and 5-COOH porphyrinogens III at histidine residues.
• 5.5. On the other hand arginine modification by 2,3-butanedione treatment altered 5-COOH porphyrinogen III decarboxylation for both enzymes. However this amino acid was not involved at the binding site of this substrate.

     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

Non-carbohydrate binding partners/domains of animal lectins

• 1.1. Protein-carbohydrate interactions are involved in a large number of biologically important recognition processes.
• 2.2. Among the participating classes of proteins lectins are defined as carbohydrate-binding proteins other than an antibody or an enzyme.
• 3.3. In addition to the essential carbohydrate-binding domain other functionally and/or structurally important sites, defined by sequence comparison or by experimental demonstration of protein-protein interactions, can be present within the lectin molecule and may be relevant for its physiological significance.
• 4.4. Sequence motifs of lectins for protein-protein interactions include amino acid structures designed for cell adhesion, growth regulatory biosignalling, intracellular routing and enzymatic activity.
• 5.5. Elucidation of the complete functional role(s) of a lectin requires accurate delineation of its carbohydrate and, if present, of its protein ligands.
• 6.6. Presence of more than one carbohydrate-binding domain in a single lectin, potential ligand properties of the glycopart of a lectin, regulatory interplay between different sites and possible interaction of complementarily shaped peptide sequences to the sugar-recognizing site should all be assessed in the quest to comprehensively explain the physiological role(s) of a lectin.

     

Stearoyl-CoA desaturase activity in primary culture of chicken hepatocytes. influence of insulin,glucocorticoid, fatty acids and cordycepin

• 1.1. Stearoyl-CoA desaturase (Δ9-desaturase) activity was measured in chicken primary hepatocytes, as a function of time in culture.
• 2.2. When using fasted donor animals, the desaturase activity was low at the beginning of culture and then increased steadily to a maximum value between 30 and 70 hr of culture. When hepatocyte cultures were prepared from fed animals, enzyme activity was high at the beginning of culture and maintained thereafter at similar values to those obtained in cultured hepatocytes from fasted animals after 30 hr of culture.
• 3.3. Insulin significantly enhanced enzyme activity when added to the culture medium at a 10⁻⁹M concentration, and a small stimulating effect was also observed with 10⁻⁶M dexamethasone.
• 4.4. Linoleic acid (0.5 mM) added to the culture medium as albuminic complex partly inhibited Δ9-desaturase activity.
• 5.5. Cordycepin (3' deoxyadenosine) decreased enzyme activity when present at a 3 μg/ml concentration in the culture medium.
• 6.6. Taken together, the induction of enzyme activity in culture, its impairment by cordycepin and response to insulin and linoleic acid strongly suggest that synthesis and translation of the Δ9-desaturase mRNA occur in chicken hepatocytes in primary culture, and that this cellular model may be a useful tool for further studies on Δ9-desaturase regulatory mechanisms.

     

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.

     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

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.

     

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.

     

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Carbonic anhydrase activity in blood of early sheep fetuses

• 1.1. The level of carbonic anhydrase activity in the red cells was measured in sheep fetuses at different times after conception: 39, 56, 77, 90 and 140 days, the last being close to full term. Measurements were also made on blood from four of the mothers.
• 2.2. There was a low level of the enzyme present in the 39 day fetuses (0.037 enzyme units (E.U.)/100 μg Hb) and its increase up to 90 days of gestation (0.19 E.U./100 μg Hb) had a form approximating exponential.
• 3.3. The earliest levels were only 11% of the full term levels and only 4% of the adult levels previously reported.
• 4.4. Even the earliest samples were of blood that was fetal rather than embryonic but these results are the earliest carbonic anhydrase activities reported in this mammal.

     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

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.

     

Amino acid substitutions within the analogous nucleotide binding loop (P-loop) of aminoglycoside 3'-phosphotransferase-II

• 1.1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme.
• 2.2. The mutations of Gly205 → Glu, Gly210 → Ala and Arg211 → Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418.
• 3.3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg²⁺ ATP.
• 4.4. Alternatively substitutions at a highly conserved basic residue (Arg211 → Lys and Arg211 → His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype.
• 5.5. Moreover, an Arg211 → His mutation drastically reduced affinity of the enzyme for Mg²⁺ ATP.
• 6.6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction.
• 7.7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a β -strand to an α-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.

     

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.

     

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 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.

     

Kinetic analysis of reversible closed bicyclic enzyme cascades covering the whole course of the reaction

A kinetic analysis of the closed bicyclic enzyme cascades is presented.

• 1.1. It includes the dependence on time from the onset of the reaction, of the concentration of the modified and unmodified enzyme species involved and the time course equations of the modificational fractions of the interconvertible enzymes.
• 2.2. The transient phase equations obtained allow the definition of new regulatory modification properties.
• 3.3. The expressions for concentrations of the unmodified and modified forms of the interconvertible enzymes, as well as those of the fractional modifications in the steady state are derived as particular cases of the general equations.
• 4.4. These steady state expressions coincide with those obtained by other authors.
• 5.5. The analytical results obtained are discussed in relation to the Escherichia coli glutamine syntethase cascade.

     

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.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.