Hydrolysis of proteins by peptide hydrolases of Antarctic krill,Euphausia superba

• 1.1. The hydrolysis of casein by peptide hydrolases of Antarctic krill, E. superba, has been
• 2.2. The peptide hydrolases studied included trypsin-like enzymes, carboxypeptidase A-type of enzymes, carboxypeptidase B-type of enzymes, and an aminopeptidase isolated from Antarctic krill.
• 3.3. The trypsin-like enzymes seemed to play a decisive role in the degradation of casein, whereas the carboxypeptidase A, carboxypeptidase B and the aminopeptidase had limited effect when acting on casein alone. When combined with the trypsin-like enzymes, the exopeptidases effected enhanced release of amino acids from the protein.
• 4.4. Based on the pattern of amino acids relased from casein by a crude extract of krill, and by the isolated peptide hydrolases either alone or in combination, it is concluded that the purified peptide hydrolases examined comprise the major enzymes responsible for the autoproteolytic activity of krill at neutral- to weakly alkaline pH.

     

Purification and characterization of carbonyl reductases from bovine liver cytosol and microsome. the cytosolic enzyme has a novel 3α/17β-hydroxysteroid dehydrogenase activity

• 1.1. Carbonyl reductase, which is distributed in both cytosolic and microsomal fractions in bovine liver, were purified to homogeneity on 12.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis and shown to have molecular weights of 32 kDa and 68 kDa, respectively.
• 2.2. Both carbonyl reductases can catalyze the reduction of many carbonyl compounds including ketone, quinones and aldehyde with relatively low K_m values.
• 3.3. From the absorption spectrum result, microsomal carbonyl reductase closely resembles cytochrome P-450 reductase.
• 4.4. Cytosolic carbonyl reductase is a novel enzyme which can act on both testosterone and androsterone at low concentration.

     

Evaluation on the hydrolysis of methylumbelliferyl-tetra-N-acetylchitotetraoside by various glucosidases. A comparative study

• 1.1. In human plasma, an enzyme is present which hydrolyzes 4-methylumbelliferyl-tetra-N-acetylchitotetraoside. The function of this enzyme is unknown.
• 2.2. We have examined whether hyaluronidase, neutral endoglucosaminidase, $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-}\text{hexosaminidase}$, aspartylglucosaminidase, β-d-glucosidase, and chitobiase could hydrolyze MU-TACT. The results obtained are detailed below.
• 3.3. A purified commercial preparation of hyaluronidase does not hydrolyze MU-TACT.
• 4.4. Substrate specificity requirements, pH optimum and subcellular localization indicate that neutral endoglucosaminidase is distinguishable from MU-TACT hydrolase. Also commercial neutral endoglucosaminidase D and H have no affinity towards MU-TACT.
• 5.5. $\text{N-}\text{Acetyl}\text{-β-}\text{d}\text{-}\text{hexosaminidase}$ is different from MU-TACT hydrolase for the following reasons: (a) a purified enzyme preparation does not hydrolyze MU-TACT; (b) there is no correlation in the activity of the enzymes; (c) MU-TACT hydrolase is not deficient in cells of a patient with a deficiency of total $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-}\text{glucosaminidase}$; and (d) the 2 enzymes have very different Chromatographic characteristics and Con A binding properties.
• 6.6. Enzyme characteristics, substrate structural requirements and a lack of correlation with MU-TACT hydrolase activity suggest that aspartylglucosaminidase, β-d-glucosidase, and chitobiase are not involved in the hydrolysis of MU-TACT.
• 7.7. None of the enzymes which we have considered corresponds to MU-TACT hydrolase. The exact nature and the function of the enzyme remains an enigma.

     

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.

     

Protein tyrosine kinase activity in cultured vascular smooth muscle cells (VSMC) from rat aorta

• 1.1. Protein tyrosine kinase (PTK) activities were detected in both cytosolic and particulate fractions of cultured vascular smooth muscle cells by using poly (Glu: Tyr; 4:1) as an exogenous substrate.
• 2.2. The percent distribution of the enzyme activity between these two fractions was 70 and 30 respectively.
• 3.3. The particulate and not the cytosolic enzyme activity was stimulated by about 4-fold in the presence of non-ionic detergent, Triton X-100 (0.5% v/v).
• 4.4. The PTK activity in both the fractions was absolutely dependent on the presence of divalent cations such as Mg²⁺ and Mn²⁺ which were equipotent in the activation of the enzyme.These data indicate that PTK activity is expressed in cultured VSMC and provide a basis for further studies to examine a possible role of PTKs in growth and proliferation of VSMC.

     

Nucleoside monophosphoramidate hydrolase from rat liver: Purification and characterization

• 1.1. Adenosine 5'-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol.
• 2.2. It consisted of two subunits of 14 kDa.
• 3.3. It hydrolyzed nucleoside 5'-monophosphoramidates into nucleoside 5'-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N^ω-phosphoarginine, 6-phospholysine and 3-phosphohistidine.
• 4.4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.

     

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.

     

Long-term starvation in Xenopus laevis daudin—III. Effects on enzymes in several tissues

• 1.1. Adult, female Xenopus laevis were subjected to 12 months of starvation.
• 2.2. Starvation resulted in a continuous reduction in the activity of both hepatic and renal glucose-6-phosphate dehydroganse.
• 3.3. Fructose-1,6-diphosphatase was significantly reduced at months 10 and 12 in the liver, and at months 4, 10, and 12 in the kidney.
• 4.4. Pyruvate kinase activity of muscle and liver decreased during the experimental period whereas the renal enzyme remained essentially unchanged.
• 5.5. Both hepatic and renal glutamate-pyruvate transaminase (GPT) and hepatic glutamate-oxaloacetate transaminase (GOT) showed a reduction of activity after 2 and 4 months of starvation followed by an increase in GPT but not in GOT.

     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

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.

     

Non-transferrin-bound-iron in serum and low-molecular-weight-iron in the liver of dietary iron-loaded rats

• 1.1. The feeding of 0.5% (3,5,5-trimethylhexanoyl)ferrocene (TMH-ferrocene) in rats resulted in a severe and progressive liver siderosis (total liver iron, 30 mg/g liver wet weight, after 30 weeks).
• 2.2. High concentrations of an iron-rich ferritin (up to 250 mg/l) were detected in serum of heavily iron-loaded rats forming a large fraction of non-transferrin-bound-iron (5000 μg/dl in maximum).
• 3.3. Ferritin and not haemosiderin was the major iron storage protein in the liver.
• 4.4. The total liver iron concentration (from 0.4 to > 30 mg Fe/g wet wt) but not the cytosolic low-molecular-weight-iron fraction (from 0.5 to 2.5 μM) was extremely increased during iron-loading.

     

The haemocytes and the activities of leucine aminopeptidase and alkaline phosphatase during development of Ceratitis capitata: Specific secretion of a leucine aminopeptidase isozyme

• 1.1. The types of haemocytes during larval development were studied.
• 2.2. The developmental profile of leucine aminopeptidase and alkaline phosphatase was studied. The maximum LAP activity was found to be in early larval development, while the maximum alkaline phosphatase during the white pupal stage.
• 3.3. These activities were compared with those determined in cell-free haemolymph.
• 4.4. Both hydrolytic enzymes have been found histochemically in the prohaemocytes and in the plasmatocytes.
• 5.5. In cultured haemocytes experiments it was found that 64% of the total LAP activity was secreted into the incubation medium, while electrophoretic analysis of released LAP activity demonstrated that only LAP A isozyme was secreted.
• 6.6. Based on the above results we suggest that both hydrolytic enzymes are functionally important throughout larval development.

     

Cellulose digestion by the firebrat Thermobia domestica

• 1.1. A highly efficient cellulose digestion could be demonstrated in a primitive insect species, Thermobia domestica (Thysanura:Lepismatidae), by the application of a uniformly ¹⁴C-labelled substrate.
• 2.2. Gut extracts exhibit distinct hydrolytic activities toward different cellulosic substrates (cellobiose, sodium carboxymethylcellulose and microcrystalline cellulose). Therefore, the complete cellular complex must be present.
• 3.3. Besides cellulases, several other carbohydrates occur in the digestive juice, thus reflecting the omnivorous feeding habits of the insect.
• 4.4. The crop was found to be the main site of carbohydrate digestiopn, also including cellulolysis.
• 5.5. It is very likely that the cellulolytic enzymes derive from the gut tissues of the firebrat.

     

Intermediary metabolism of normal and tumorous tissue of Xiphophorus (Teleostei: Poeciliidae)

• 1.1. The Xiphophorus melanoma system is one of the few well established and genetically well understood in vivo models in experimental carcinogenesis. However, data describing features of intermediary metabolism of the genetically caused melanoma or the different inducible neoplasia, as well as that of the different transformed cell lines of Xiphophorus, are still lacking. For this reason we initiated a comparative study of enolase-, pyruvate kinase-, lactate dehydrogenase- and malate dehydrogenase-activities and pyruvate and lactate levels in transformed as well as normal tissues of Xiphophorus.
• 2.2. We observed tissue specific and age dependent activities of the different enzymes and substrate levels.
• 3.3. Enzyme activities and substrate levels from all tumors analyzed differ from that of any normal tissue. They are dependent on the tumor sections analyzed, the histiotype and the etiology of the tumors.
• 4.4. Analysis of enzyme activities from different in vitro cultured fish cell lines and the human Hela cell line revealed dependency of the intermediary metabolism on oxygen supply, on the proliferative state of the cells and on the cell types.
• 5.5. We could not find a correlation between our data and the expression of the c-src gene of Xiphophorus and no genotype-dependent changes in enzyme activities were detected.

     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

Gender-related differences of mouse liver d-aspartate oxidase in the activity and response to administration of d-aspartate and peroxisome proliferators

• 1.1. The hepatic d-aspartate oxidase activity was found to be higher in female ddY and ICR mice than in their male counterparts. On the contrary, the free d-aspartate content in the liver was lower in female mice than in male mice, suggesting that d-aspartate is actually metabolized by d-aspartate oxidase in vivo.
• 2.2. Oral administration of d-aspartate to the animals increased the hepatic d-aspartate oxidase activity 2–3 fold in both genders without any significant difference in the rate of the increase between the genders.
• 3.3. Several peroxisomal enzyme activities other than d-aspartate oxidase examined were not affected by this treatment.
• 4.4. Experiments in vitro suggested that the increase in the d-aspartate activity might be explained in part by stabilization of the enzyme by d-aspartate.
• 5.5. The administration of clofibrate, a peroxisome proliferator, to male mice, increased the hepatic d-aspartate oxidase activity with a significant simultaneous decrease of d-aspartate content in the liver, in agreement with a possible role of the enzyme n vivo.
• 6.6. On the other hand, the administration of clofibrate or dehydroepiandrosterone to female mice decreased the d-aspartate oxidase activity.
• 7.7. The peroxisome proliferators were suggested to act to eliminate the gender difference of hepatic d-aspartate oxidase activity in mice.

     

Dipeptidyl aminopeptidase activities of guinea-pig brain

• 1.1. The subcellular distribution ofdipeptidyl aminopeptidase activities in guinea-pig brain was investigated. Our studies show that DAP I (Gly-Arg-NH-Mec hydrolase) type activity was found to have an acidic optimum and was associated with the nuclear pellet.
• 2.2. No DAP II (Lys-Ala-NH-Mec hydrolase) type activity could be detected. Apparant hydrolysis was mainly due to aminopeptidase activity.
• 3.3. DAP III (Arg-Arg-NH-Mec hydrolase) type activity is largely cytoplasmic, but there was evidence of a membrane form associated with the synaptosomes.
• 4.4. DAP IV (Gly-Pro-NH-Mec hydrolase) type activity is present on the synaptosomal membrane, and also enriched in the microsomes. A soluble form of Gly-Pro-NH-Mec hydrolase activity is also present in the cytoplasm. Whether this activity is a DAP II or IV type activity is still yet to be determined.

     

Protein tyrosine phosphorylation in normal rat tissues

• 1.1. Exogenous and endogenous tyrosine protein phosphorylation activities were examined in soluble and partieulate fractions from various normal tissues by using poly-[Glu-⁸⁰Na, Tyr²⁰] and a monoclonal antibody specific for phosphotyrosine.
• 2.2. Phosphorylation of the exogenous substrate by the partieulate forms of TPKs was 2- to 10-fold higher than by soluble forms. The activities of partieulate and soluble enzymes decreased in the following order: spleen > (thymus = kidney) > testes ⩾ (pancreas = liver = brain) > heart.
• 3.3. The level of endogenous phosphorylation in the tissues decreased respectively in the following order: thymus > brain ⩾ (pancreas = liver) > spleen > testes > kidney > heart for the partieulate fractions, and spleen > thymus > brain > pancreas ⩾ liver > testes > kidney > heart for the soluble fractions.
• 4.4. A large number of phosphotyrosine-containing proteins were detected. In addition, several phosphotyrosine-containing proteins of similar molecular weight were found in different tissues and fractions.

     

Influence of handling and/or anaesthesia on stress response in rainbow trout. Effects on liver primary metabolism

• 1.1. The overall effect of handling, anaesthesia and sham injection on some blood metabolites, liver glycogen and several key enzymes involved in liver carbohydrates and nitrogen metabolism was studied in rainbow trout. In addition, the possible role of anaesthesia (MS222) itself as a stress-inductor or suppressor was also studied.
• 2.2. Stress resulted in hyperglycaemia and initially in liver glycogen depletion, as well as increasing plasma amino acid levels.
• 3.3. Glycogen stores subsequently recovered while amino acid concentration fell.
• 4.4. These changes seemed to correlate with the increased activity of liver fructose 1,6-bisphosphatase, glucose 6-phosphate dehydrogenase, alanine aminotransferase and glutamate dehydrogenase, thus supporting the hypothesis that gluconeogenic flux from amino acids increases in stressed trouts.
• 5.5. Anaesthesia, under the same experimental conditions, did not seem to mediate in stress production, but rather resulted in stress suppression.