Identification of cathepsin L-like proteinase and aminopeptidase in yolk granules of the sand worm,Nereis diversicolor

• 1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.
• 2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.
• 3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.
• 4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.

     

On the purification and characterization of exopeptidases from antarctic krill,Euphausia superba

• 1.1. Two carboxypeptidase-A type of enzymes and two carboxypeptidase-B type of enzymes effecting hydrolysis of Hipp-l-Phe and Hipp-l-Arg respectively, have been purified from E. superba using gel filtration, affinity chromatography and FPLC-anion exchange chromatography. In addition an aminopeptidase has been partly purified.
• 2.2. The carboxypeptidases had mol. wts of 27,000 (carboxypeptidase A) and 31,000 (carboxypeptidase B).
• 3.3. Carboxypeptidase A exhibited a broad pH optimum with a maximum at pH 5.5–6.5, whereas carboxypeptidase B had a more narrow pH-optimum with a maximum at pH 7. The aminopeptidase had an optimum at about pH 8.7.
• 4.4. The carboxypeptidases were inhibited by the chelating agent 1,10-phenanthroline.

     

The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii)

• 1.1. Oxygen equilibrium curves were measured on trout red blood cell suspensions at pH 7.8 and 8.4 at 15, 20 and 25 C. Normal red cells and red cells that had been depleted of their ATP content were used.
• 2.2. The equilibrium data were fitted to the Adair's model and the enthalpy (ΔH) and entropy (ΔS) changes for the first and fourth steps of oxygenation and for overall oxygenation were calculated from the temperature dependencies of the Adair constants.
• 3.3. For normal red blood cells, the apparent heat for the first oxygenation step, δh₁, is close to zero.
• 4.4. Temperature insensitivity of this step at physiological pH, combined with a large pH dependence, probably denotes a property of Hb4, the Root effect Hb of trout blood.
• 5.5. At pH 7.8, ΔH₄ is about —4kcal/mol, a small value which may be attributed to the large release of Bohr protons that occurs at the last oxygenation step and corresponds to an endothermic process which opposes to the exothermic oxygenation of the haem.
• 6.6. The ΔH₄ value appears to have a large influence on the enthalpy for overall oxygenation.
• 7.7. Results for ATP-free red cells are consistent with a mere increase in the intracellular pH and suggest that ATP has no specific effect at and above pH_i ~ 7.7.
• 8.8. Effects of temperature and pH on trout red blood cell isotherms emphasize the primary importance of the major component of trout blood, namely Hb4, in trout blood functional properties.

     

Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus

• 1.1. Spore coat extracts from Bacillus thuringiensis subspecies kurstaki and israelensis and Bacillus cereus T and B. cereus NRRL 569 were characterized by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by amino acid analysis.
• 2.2. Both B. cereus spore coats had similar electrophoretic profiles.
• 3.3. The B. thuringiensis spore coats contained crystal proteins as major components as well as lower mol. wt proteins.
• 4.4. B. thuringiensis subsp. israelensis had a unique coat protein profile which was different from B. cereus and B. thuringiensis subsp. kurstaki coats.
• 5.5. Insecticidal activity of spores against the tobacco hornworm, Manduca sexta, and the mosquito, Aedes aegypti, also was determined.
• 6.6. B. thuringiensis subsp. kurstaki spores were lethally toxic to the tobacco hornworm (Lepidoptera) larvae, whereas spores of the other subspecies were not.
• 7.7. Except for subspecies israelensis, none of the spores was effective against the mosquito (Diptera) larvae.

     

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.

     

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.

     

Digestive proteases in the midgut gland of the atlantic blue crab,Callinectes sapidus

• 1.1. Digestive proteases from the midgut gland of male Atlantic blue crabs, Callinectes sapidus, were investigated. Tentative identities of proteolytic enzymes were determined with synthetic substrates and inhibitors.
• 2.2. Trypsin, chymotrypsin, carboxypeptidase A and B and leucine aminopeptidase activities were found and quantified.
• 3.3. Activity against Succinyl-(Ala)₃-nitroanalide was also found. This as yet unidentified enzyme has a mol. wt of about 26,000 and has elastolytic activity.

     

Changes in composition and proteolytic enzyme activities of artemia during early development

• 1.1. The proximate composition, total and free amino acids, and proteases of Artemia nauplii were determined during early development.
• 2.2. Moisture increased from 71.0% to 80.8%, crude protein decreased from 13.2% to 8.8%, crude fat and ash varied slightly.
• 3.3. The total amino acids decreased. Free amino acids changed in three patterns.
• 4.4. Trypsin, chymotrypsin, carboxypeptidase A, B and cathepsin B and C increased in activity. The activity of trypsin was lower, while cathepsin B and C were the highest.
• 5.5. The protease activities were maximal at pH 7.5 and 8.0, and at 45°C on casein.
• 6.6. The optimal pH for carboxypeptidase A was 4.0, for carboxypeptidase B was 4.5, for trypsin and chymotrypsin were 7.0–7.5. The protease(s) active at pH 9.0–9.5 were to be determined.

     

Effects of membrane-perturbing drugs and noradrenalin on cAMP accumulation and red cell water content in carp (Cyprinus carpio) red cells

• 1.1. Carp red cells were treated with drugs that affect the cell membranes. The water content of the cells and the accumulation of cAMP in the cells were measured in normoxia and in hypoxia using non-stimulated and adrenergically stimulated cells.
• 2.2. WGA, DIDS + CCCP and A23187 increased the water content of nonstimulated normoxic cells.
• 3.3. In hypoxia ouabain and DIDS + CCCP increased the water content but cytochalasin B, NPM, DIDS, CCCP and A23187 + CA²⁺ abolished the hypoxia-induced swelling.
• 4.4. Any membrane perturbation induced some cAMP formation, Sophora and Anquilla lectins being most potent.
• 5.5. Also in adrenergically stimulated cells, membrane perturbation generally increased cAMP formation.
• 6.6. However, cAMP accumulation diminished in cells treated with cytochalasin B, CCCP and DIDS + CCCP.
• 7.7. The adrenergic swelling of carp red cells was reduced in normoxia by DIDS. NPM and CCCP increased the adrenergic swelling in normoxia to hypoxic level.
• 8.8. In hypoxia WGA and Anquilla lectin decreased the swelling.

     

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.

     

Blood values in South American lizards from high and low altitudes

• 1.1. Blood indices were measured in 27 species of lizard from Chile and Argentina occurring at different altitudes ranging from sea-level up to 4600 m.
• 2.2. Contrary to amphibians, none of the hematological values of these lizards, such as hematocrit, hemoglobin concentration, red cell count, mean cell volume, mean cell hemoglobin and mean cell hemoglobin concentration, were found to be correlated with their altitudinal distribution.
• 3.3. Intrageneric comparison of blood values in Liolaemus lizards (seven highland species living above 3000m and 12 lowland species) showed a similar degree of independence from their altitudinal site of capture or from their upper limit of distribution.
• 4.4. As reported for other vertebrate taxa, an inverse correlation between size and number of red blood cells was also found in the studied reptiles.

     

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.

     

Hematology and blood chemistry of the marsh harrier (Circus aeruginosus)

• 1.1. This study deals with the hematological and blood chemistry of 13 adult marsh harriers (Circus aeruginosus).
• 2.2. No significant differences were observed between male and female groups in any of the parameters.
• 3.3. The value of white blood cells was 14,677/mm³ heterophils and lymphocytes, these being the most abundant cellular type (81.42 and 12%, respectively).
• 4.4. Urea and uric acid are present in approximately similar proportions, though birds are said to be uricotelic.
• 5.5. The cholesterol concentration values determined in our study, are higher than those reported in most other birds.

     

Trypanosoma cruzi and Trypanosoma rangeli: Glutamate dehydrogenases and proteolytic activities

• 1.1. Trypanosoma (Herpetosoma) rangeli contains proteolytic activity with azocasein, casein and BAPA as substrates, and aminopeptidase activity with Arg-BNA as substrate. The respective pH optima were 5.5, 7.0, 8.5 and 7.0.
• 2.2. The effect of the protease inhibitors PMSF, TLCK and trasylol was studied. 0.5 mM TLCK caused considerable inhibition of all these activities, whereas 1 mM PMSF was much less effective. Trasylol (0.14 mg/ml) inhibited the activities with azocasein and Arg-BNA as substrates.
• 3.3. Trypanosoma (Schizotrypanum) cruzi contains very similar proteolytic activities, with some slight differences in pH optima and in response to inhibitors. Thus Trasylol was not effective on any activity, and the activity on Arg-BNA was little sensitive to TLCK.
• 4.4. The levels of these enzymes, and also of the NAD- and NADP-linked glutamate dehydrogenases, were studied in six stocks of T. (Sch.) cruzi and three stocks of T. (H.) rangeli. There was no significant difference in the NAD-gluDH, nor in the proteolytic activity with BAPA. On the other hand, the other enzymes tested presented differences which ranged from about 3-fold for the aminopeptidase to nearly 100-fold for the NADP-gluDH. The electrophoretic behaviour of the latter was identical in all the stocks of both species, thus showing that the difference was only quantitative.
• 5.5. The three stocks of T. (H.) rangeli were more similar to some T. (Sch.) cruzi stocks (Tul 2) than the latter were to each other, thus emphasizing at a biochemical level the similarities between these two species.

     

Seasonal changes of circulating blood parameters in the grass snake Natrix natrix natrix L.

• 1.1. Seasonal changes of circulating blood parameters of Natrix n. natrix were evident and involved both sexes to the same extent.
• 2.2. A significant decrease in red cell count, haematocrit and haemaglobin concentration in the mating period, and an increase in those parameters and mean cell volume in autumn were observed, and haemodilution during winter torpor.
• 3.3. The changes during the breeding season had probably a hormonal background; in winter, they resulted first of all from a decreased erythropoietic activity and, to a lesser extent, from an increased red blood cell breakdown rate. However, the possibility that some erythrocytes were withdrawn from the circulation cannot be excluded.
• 4.4. Winter lymphocytopenia, eosinocytopenia and neutrophilic granulocytosis in females during egg laying were expressions of changes of leucocyte formula.
• 5.5. Seasonal cyclicity was found only with respect to the white cell count in males and the eosinophile fraction in males and females.
• 6.6. Probable reasons for, and mechanisms of the changes in blood composition are discussed.

     

The effect of reduced salinity on tissue and plasma composition of the dogfish,Squalus acanthias

• 1.1. Dogfish (Squalus acanthias) were acclimated to reduced salinities and their plasma, muscle tissue and erythrocytes subsequently analysed.
• 2.2. Decrease in the osmolarity of the plasma was principally due to a fall in urea concentration and a significant fall in the concentrations of sodium and chloride.
• 3.3. Changes in the muscle and erythrocytes in dilute media were a decrease in urea, potassium, sodium and chloride concentrations.
• 4.4. The concentrations of the free amino acids in the muscle and the red blood cells decreased more than would be expected by the movements of water only.
• 5.5. The results were discussed in relation to the regulation of cellular volume and the involvement of the free amino acid pool of the tissues in this process.

     

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.

     

Lipid composition of the red and green forms of Actina equinia from the black sea

• 1.1. The fatty-acid composition of the red and green forms of Actinia equina from the Black Sea have been determined by methods involving silver-ion HPLC and GC-MS.
• 2.2. The fatty acid compositions of both forms of A. equina resemble those of most marine invertebrates. Substantial amounts of C₂₀ and C₂₂ polyunsaturated fatty acids were found.
• 3.3. Balck Sea Actenia equina contains a large amount of plasmalogens, mainly phospotidylcholine and phosphatidylserine plasmalogens.
• 4.4. The red form of A. equina contains more arachidonic acid in glycolipid fraction and more phosphatidylethanolamine and its plasmalogen, while the green from contains more sphingomyelin. These differences are an indication that the species A. equina can be divided into two subspecies—green and red.

     

Structural features of carbohydrate chains in human salivary mucins

• 1.1. The structure of carbohydrate chains in the low and high molecular weight mucus glycoprotein forms from submandibular-sublingual saliva of individuals with blood group B was investigated.
• 2.2. Alkaline borohydride reductive cleavage of the glycoproteins yielded in each case a population of neutral (55%) and acidic (45%) oligosaccharide alditols ranging in size from 3 to 16 sugar units.
• 3.3. The predominant neutral oligosaccharides in both glycoprotein forms consisted of 16 and 15 sugar units arranged in triantennary fashion, and carried blood group B and I antigenic determinants.
• 4.4. Three of the oligosaccharides in each glycoprotein contained sialic acid and ranged in size from 3 to 12 sugar units. In two oligosaccharides sialic acid was linked to C3 of galactose and in one to C6 of N-acetylgalactosamine. The sulfated oligosaccharide in both glycoproteins was identified as a pentasaccharide with the sulfate ester group at C6 of N-acetylglucosamine.
• 5.5. The results demonstrate that contrary to the earlier view the low and high molecular weight mucus glycoprotein forms of human saliva contain identical carbohydrate chains.

     

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.