Characterization of two distinct latent proteinases associated with myofibrils of crucian carp (Carassius auratus cuvieri)

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• 1.1. Enzymatic properties of two distinct proteinases tightly associated with crucian carp myofibrils were characterized.
• 2.2. These proteinases were latent but activated at 50 and 60°C, respectively.
• 3.3. The optimum pH of 50°C-proteinase was neutral-alkaline, while that of 60°C-proteinase was weak acid-neutral pH.
• 4.4. Both proteinases required more than 1% NaCl for the activity, but 50°C-proteinase was partially inhibited at higher concentrations of NaCl.
• 5.5. Both proteinases were regarded as trypsin-like proteinases belonging to a serine proteinase family, but only 60°C-proteinase was sensitive to urea, n-butanol and iso-propanol.

     

Collagen diversity in the sea urchin,strongylocentrotus purpuratus

• 1.1. Pepsin insensitive fragments of collalgen extracted from tube feet and peristome have α 1 and α 2 bands that differ in apparent molecular weights from each other and from human type 1 collagen.
• 2.2. A monoclonal antibody that reacts with the ξ I band and a low molecular weight fragment of tube foot collagen does not react with either peristome collagen or human type I collagen.
• 3.3. Measurements of the axial periodicity of native fibers of tube foot and peristone collagens indicate they have D values that differ significantly from each other and from reported values of vertebrate type I collagen.
• 4.4. We propose that there are diverse and specialized types of collagen in sea urchins that are heterogeneously distributed in the extracellular matrix of different tissue.

     

Tissue specific isoenzymes of d-lactate dehydrogenase from the foot,mantle and hepatopancreas of Patella caerulea (L). purification and properties

• 1.1. Isoenzymes of d-lactate specific dehydrogenase from foot, mantle and hepatopancreas of Patella caerulea have been purified by Chromatographic techniques. d-lactate dehydrogenase (d-Ldh) from P. caerulea tissues was found to be tetrameric with a M_r of ca 140,000 as judged by gel filtration; subunit M_r of ca 37,000 was obtained from SDS-electrophoresis.
• 2.2. Kinetic studies suggest that P. caerulea foot and mantle d-Ldh is similar to vertebrate muscle-type l-Ldh; furthermore hepatopancreas d-LDH resembles vertebrate heart-type l-LDH since it has a higher affinity for d-lactate and is inhibited by pyruvate.
• 3.3. The results imply that the P. caerulead-Ldh isoenzymes may have distinct metabolic functions.

     

AMP-deaminase from human skeletal muscle. Subunit structure,amino-acid composition and metal content of the homogenous enzyme

• 1.1. Homogenous human skeletal muscle AMP-deaminase was obtained by chromatography on phosphocellulose.
• 2.2. Native enzyme molecular weight was 290,000, while a value of 71,000 was found for the subunit molecular weight.
• 3.3. No distinct differences were found in amino-acid composition of human skeletal muscle AMP-deaminase as compared with other vertebrate enzymes.
• 4.4. Human muscle AMP-deaminase contains about 2g-atom of zinc per 280,000; considerable amounts of calcium and magnesium were also found.

     

Content and partial characterization of collagen in crustacean muscle

• 1.1. The collagen content in the abdominal muscle of seven species including shrimp, prawn, lobster and squilla varied among the species ranging from 1.1 to 6.2% of total tissue protein and the content in pereiopod and thoracic muscles of four species of crab varied ranging from 0.2 to 0.8%.
• 2.2. These results indicate that the musculature in flexible part comprises a high proportion of collagen.
• 3.3. The major collagen from the crustacean muscle was found to be similar to Type V collagen from the vertebrate muscle with respect to the solubility and amino acid composition.

     

S-Adenosyl-l-methionine decarboxylase activities in Mytilus edulis

• 1.1. The activities of S-adenosylmethionine decarboxylase (EC 4.1.1.50) were measured in cell extracts of mantle, hepatopancreas and foot from Mytilus edulis.
• 2.2. The apparent molecular weights of the enzymes estimated by gel filtration chromatography were 65,000 ± 10,000.
• 3.3. The enzymes do not require bivalent cations for catalysis and show optimum pH between 7.0–8.0 in phosphate buffer.
• 4.4. The hepatopancreas enzyme shows different behavior to the other two enzymes against temperature and its activity is strongly inhibited by NH₄⁺.
• 5.5. The apparent K_ms for S-adenosylmethionine were found to be 300, 200 and 250 μM for the hepatopancreas, mantle and foot enzymes, respectively.

     

Characterisation of the electron transport chain of an obligate methylotroph,strain 4025

• 1.1. The obligate methanol-utilising bacterium strain 4025 contains cytochromes b and c. Cytochrome a is never present.
• 2.2. The soluble cytochrome c is similar to that from other methylotrophs in reacting (slowly) with carbon monoxide and it can be separated into two types, differing markedly in their isoelectric points.
• 3.3. Some of the cytochrome b reacts rapidly with carbon monoxide and is thus the likely cytochrome oxidase (cytochrome o).
• 4.4. The partially purified, NAD⁺-independent methanol dehydrogenase is similar to such enzymes from the other methanol-utilising bacteria in respect of its prosthetic group, dependence on ammonia or methylamine for activity and its wide substrate specificity.
• 5.5. The fluorescence seen in colonies of this organism is probably due to a flavin derivative.
• 6.6. This study of electron transport components does not shed any light on the unusually high copper requirement shown by this methylotroph.

     

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.

     

Identification of proteolytic isozymes from Antarctic krill (Euphausia superba) in an enzymatic debrider

• 1.1. A partially purified krill extract (enzymatic debrider) intended for clinical use was electrophoretically characterized by polyacrylamide gel electrophoresis (PAGE) and by crossed immunoelectrophoresis (CIE) using polyclonal rabbit antibodies.
• 2.2. Three main types of proteolytic enzymes (serine proteinase, carboxypeptidase A and B) with mol. wts of 33,000, 28,000 and 35,000, respectively, could be separated by SDS—g-PAGE under reducing conditions.
• 3.3. Routine CIE analysis of krill samples revealed four protease-active immunoprecipitates. Two of these precipitates were associated with the proteinase activity, one with carboxypeptidase A and one with carboxypeptidase B.
• 4.4. Improving resolution of CIE by extending electrophoresis in the first dimension permitted separation of three serine proteinases of which two were isozymes (II and III) and the third one was unique (I).
• 5.5. Furthermore carboxypeptidase A could also be separated into two isozymes (AI and AII) while carboxypeptidase B still exhibited one single component.
• 6.6. Six individual immunoprecipitates were thus identified and proved to be related to the protease activity. Highly purified enzymes were used as references in CIE and tandem-CIE to establish identification of each enzyme in the krill mixture.

     

Characterization of diverse hemolymph lectins in the cotton caterpillar Spodoptera littoralis

• 1.1. Hemolymph lectins (agglutinins) of the cotton caterpillar Spodoptera littoralis were analyzed by agglutination, cross-absorption and carbohydrate-hemagglutination inhibition using several vertebrate erythrocytes.
• 2.2. Lectins were found to interact, with all tested erythrocytes, by binding to carbohydrate moieties but showing no definite specificity.
• 3.3. Disulphide bonds were probably absent as 2-ME treatment was ineffective.
• 4.4. By cross-absorption studies, we have proposed that the hemolymph contains multiple lectins.

     

Hydrolysis of glycol chitin by chitinolytic enzymes

• 1.1. The hydrolysis of glycol chitin preparations by several β-N-acetylglucosaminidases was monitored colorimetrically with the potassium ferriferrocyanide reagent.
• 2.2. Glycol chitin samples from crab and insect sources varied considerably in chemical composition and susceptibility to enzymatic hydrolysis.
• 3.3. Insect endochitinase preferred crab glycol chitin as substrate while hen's egg white lysozyme preferred commercial glycol chitin.
• 4.4. Insect glycol chitin was well hydrolyzed by both enzymes.
• 5.5. Insect exochitinase did not digest glycol chitin.

     

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.

     

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.

     

Reducing equivalent transport by substrate shuttles in rat liver and colon

• 1.1. The observed level and subcellular distribution of the α-glycerophosphate and malate-aspartate substrate shuttle enzymes in liver and colon were consistent with their proposed roles in reducing equivalent transport.
• 2.2. K_m value determinations of shuttle enzymes were performed.
• 3.3. Substrate shuttles were reconstructed from isolated liver and colon mitochondria which displayed satisfactory respiratory control and P:O ratios.
• 4.4. The results obtained suggest that while the malate-aspartate shuttle is the primary means of reducing equivalent transport in the liver, the α-glycerophosphate shuttle predominates in the colon.

     

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.

     

Locust vitellogenin receptor: an acidic glycoprotein with N- and O-linked oligosaccharides

• 1.1. The locust vitellogenin (VTG) receptor which is embedded in oocyte plasma membranes is a glycoprotein.
• 2.2. With various lectins oligosaccharide units have been identified, among them neuraminic acid linked to Gal or GalNAc, mannose chains, Gal linked to GalNAc or GlcNAc and fucose linked to GlcNAc.
• 3.3. With specific enzymes it could be shown that mannose and most other oligosaccharides are O-linked while others like fucose are N-linked.
• 4.4. Enzymatic removal of all O-linked carbohydrates resulted in a drop of the molecular mass of the receptor protein from 200,000 to 110,000.
• 5.5. A total of N- and O-linked oligosaccharides of 54% was calculated.
• 6.6. The isoelectric point of the receptor was found to be at pH 3.4 increasing slightly after removal of neuraminic acid.
• 7.7. Removal of neuraminic acids destroyed the binding ability for VTG.

     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

Possibilities of biochemical taxonomy of bats using hemoglobin,lactate dehydrogenase,esterases and other proteins

• 1.1. Bat hemoglobin resembles other mammalian Hb's in its physiological properties, and Hb differences among bat species are minor.
• 2.2. One polymorphism of the H chain of lactate dehydrogenase occurs in Myotis lucifugus and M. keenii, and another occurs in Eptesicus fuscus. Bat heart and muscle have identical LDH isozyme profiles.
• 3.3. Esterases and major low ionic strength extractabe proteins show a number of differences at the generic level, as well as some polymorphisms which cross species lines.
• 4.4. The protein studies indicate that bats have great individual variation, often of a type comparable to known genetically based protein polymorphisms in other species, but apparently have not had time to accumulate extensive divergent species specificity.

     

Collagenaceous,thiol-containing proteins of cnidarian nematocysts: A comparison of the chemistry and protein distribution patterns in two types of cnidae

• 1.1. Nematocyst structural proteins (NSP) from the sea anemones Aiptasia pallida and Metridium senile and the siphonophore Physalia physalis are primarily low molecular weight collagens linked by disulfide bonds.
• 2.2. NSP patterns resolved by SDS-PAGE revealed a common, major collagen species (40 kDa) in each nematocyst type, together with other collagens and non-thiol-containing proteins.
• 3.3. For each cnidarian, NSP glycosylation profiles were significantly different.
• 4.4. Monoclonal antibodies against Aiptasia NSP demonstrated a differential distribution between capsule wall and thread.
• 5.5. NSP differences would account for the diversity of morphologic and functional types.