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.

     

Ostrich α-amylase: Purification and characterization of the pancreatic isoenzymes

• 1.1. Four ostrich pancreatic α-amylase isoenzymes were isolated by isoelectric focusing, following affinity chromatography on cyclohepta-amylose-Sepharose 4B.
• 2.2. Amino acid compositions of the four isoenzymes are very similar with only one charged amino acid (Arg) being significantly different.
• 3.3. The molecular weights, as determined by SDS-PAGE and amino acid composition, are nearly identical (52–53 kDa) for all four isoenzymes.
• 4.4. The four α-amylase isoenzymes appear to be kinetically distinct enzymes with a requirement for calcium.
• 5.5. Ostrich α-amylase isoenzymes appear to be non-glycosylated and contain one free thiol group.

     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

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.

     

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 yolk polypeptides of a free-living rhabditid nematode

• 1.1. The yolk proteins of hermaphrodite Dolichorhabditis sp. (Nematode, Rhabditida) are composed of at least three polypeptides: VT1, VT2 and VT3 with molecular masses of 175.2, 107 and 82 kDa respectively.
• 2.2. All three yolk polypeptides make up at least one native protein complex which can be resolved by PAGE.
• 3.3. The yolk proteins are glycosylated and can be isolated by chromatography in Con A-Sepharose.
• 4.4. Partial chymotryptic hydrolysis shows that VT2 in different from its C. elegans homologue, YP115.
• 5.5. The main polypeptides synthesized by whole animals are the yolk components which are actively secreted in the incubation medium.

     

Evidence for a molecular polymorphis of cholinesterase in Sepia officinalis (cephalopoda: decapoda)

• 1.1. Three forms of cholinesterase were sequentially extracted from head and tentacles of Sepia officinalis and noted as low-salt (LSS), detergent (DS) and high-salt (HSS) soluble. They represent about 24, 30 and 46% of total activity.
• 2.2. All enzyme forms seem to be amphiphilic proteins with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation (LSS form).
• 3.3. The DS form is membrane-anchored by a phosphatidylinositol, while the HSS form is likely linked to some proteoglycan molecule of the extracellular matrix by ionic interactions.
• 4.4. According to V_max/K_m values, all the enzymes are acetylcholinesterases, even if hydrolyze propionylthiocoline at the highest rate.
• 5.5. Some kinetic and molecular properties of the studied enzymes are compared with those of other cholinesterases from vertebrates and invertebrates. Possible phylogenic and adaptive features are discussed.

     

Neurohypophysial hormones as molecular evolutionary tracers: investigations on the sturgeons Acipenser stellatus and Acipenser guldenstadti

• 1.1. Neurohypophysial hormones of two sturgeon species, Acipenser stellatus and Acipenser guldenstadti, have been purified through molecular sieving on Bio-Gel P4 and reverse-phase high pressure liquid chromatography on Nucleosil C18 columns.
• 2.2. Arginine vasotocin has been identified in both species by its retention time in partition chromatography, amino acid composition and, in the case of A. stellatus, by amino acid sequencing.
• 3.3. A second peptide has been purified and could be α-deamidated vasotocin.
• 4.4. Another peptide with oxytocic activity, distinct from the known oxytocin-like peptides, seems to be present in very small amounts.

     

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.

     

Purification and some properties of isocitrate dehydrogenase of a blowfly Aldrichina grahami

• 1.1. NADH-dependent isocitrate dehydrogenase has been purified 110-fold from the crude extract of the flight muscle mitochondria of Aldrichina grahami.
• 2.2. The purification procedure involved Triton X-100 treatment of isolated mitochondria, column chromatography on DEAE-cellulose, Affi-gel blue, and P-cellulose.
• 3.3. The purified enzyme was homogeneous by criteria of the polyacrylamide gel electrophoresis.
• 4.4. The enzyme of the blowfly contains more acidic amino acids and less hydrophobic amino acids than that of pig heart.
• 5.5. The molecular weight was determined to be 330,000 daltons. The subunit construction differs from ghat of mammalian isocitrate dehydrogenase.

     

Digestive protease,lipase and amylase activities in stage i larvae of the american lobster,Homarus americanus

• 1.1. Digestive protease, lipase, and amylase of Stage I larvae of the American lobster Homarus americanus are characterized.
• 2.2. A sensitive method for detection of crustacean lipase was developed using an latroscan which combines thin-layer chromatography and flame ionization detection to quantify free fatty acids generated by lipase digestion.
• 3.3. pH optima of the three enzymes occurred at or near the pH of gastric fluid.
• 4.4. A time course study demonstrated slight increases in protease and amylase activities during the first larval stage, regardless of whether the lobsters were fed or not, whereas lipase activity was constant.

     

Multiple hemoglobins in Triturus cristatus: their study by analytical isoelectrofocusing

• 1.1. Electrophoretic separation of the hemoglobin of healthy adult Triturus cristatus reveals four components.
• 2.2. Isoelectric focusing of the samee hemolysates in various commercial ampholytes of different chemical composition and pH range results in the separation of eight individual hemoglobin bands.
• 3.3. The bands obtained by electrophoresis are not homogeneous as revealed by individual gel electrofocusing. They finally separate into the same eight components, as in the whole hemolysate.
• 4.4. From the above findings it is concluded that this species has not four but eight individual hemoglobin molecular forms.
• 5.5. Our results demonstrate lack of hemoglobin polymorphism in this species.

     

Collagenolytic enzymes from the starfish,Pycnopodia helianthoides

• 1.1. Two collagenolytic proteinases have been isolated from the starfish, Pycnopodia helianthoides and partially characterized.
• 2.2. The larger of these two enzymes, with a molecular weight of approx 60,000, resembles the vertebrate collagenases in that it is inhibited by ethylenediamine tetraacetate and is able to cleave the collagen triple-helix.
• 3.3. The smaller enzyme, with a mol. wt of approximately 16,500, resembles the vertebrate chymotrypsins in its ability to hydrolyse acetyl-tyrosine-ethylester and its cleavage of collagen in the telopeptide region.
• 4.4. Other properties of the enzymes, including alkaline pH optima, acidic isoelectric point and heat stability are similar for both proteinases.

     

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.

     

Purification of two lipases with high phospholipase A1 activity from guinea-pig pancreas

• 1.1. Two cationic lipases (Ia and Ib) were purified from homogenates of fresh guinea-pig pancreas by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose (twice for the latter) followed by gel filtration on Sephadex G-100.
• 2.2. Both enzymes were homogeneous upon polyacrylamide gel electrophoresis. Their molecular weights are 37000 and 42000 for lipases Ia and Ib, respectively, as determined by gel filtration on Sephadex G-100. Very close values for isoelectric points were found in the pH range 9.3–9.4.
• 3.3. The cationic lipases are characterized by a high phospholipase A activity (500 IU/mg protein using a potentiometric assay with egg yolk lecithin as substrate), resulting in an unusual phospholipase/lipase activity ratio of 1.
• 4.4. Using doubly labelled phosphatidylcholine, a specificity, A₁, was described for the two enzymes, which are unaffected by N-ethylmaleimide, diisopropylfluorophosphate and p-bromophenacylbromide. The enzymes are insensitive to EDTA and slightly inhibited by CaCl₂and MgCl₂, whereas sodium deoxycholate is required for maximal activity.

     

Improved isolation and further characterization of beta-trichosanthin,a ribosome-inactivating and abortifacient protein from tubers of trichosanthes cucumeroides (cucurbitaceae)

• 1.1. Beta-trichosanthin was isolated from root tubers of Trichosanthes cucumeroides with a procedure involving acetone fractionation, ion exchange chromatography on CM-Sepharose and DEAE-Sepharose and gel filtration on Sephadex G-50.
• 2.2. The protein was homogeneous by SDS-polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. It possessed a molecular weight of 28,000 and was a strongly basic glycoprotein.
• 3.3. It was immunochemically identical to trichosanthin but different from alpha- and beta-momorcharins.
• 4.4. It possessed potent abortifacient and ribosome-inactivating activities. In the latter type of activity it was more potent than trichosanthin.

     

Fatty acid composition of depot fats,and rumen wall of the camel (Camelus dromedarius

• 1.1. Fatty acid composition of depot fats (hump, subcutaneous, mesentry, abdomen, perinephric and peticardiac) and rumen wall of Camelus dromedarius were studied by gas-liquid chromatography.
• 2.2. All depot fats showed similar characteristics; major fatty acids found were palmitic, stearic, oleic and myristic. Small quantities of odd-numbered, palmitoleic, linoleic and linolenic acids were also present.
• 3.3. Fatty acid of rumen wall showed some differences from the general pattern.
• 4.4. Fatty acid composition of the camel is discussed in relation to that of other ruminants and monogastrics.

     

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.

     

Human-induced tachycardia in wild and tame mallard (Anas platyrhynchos)

• 1.1. The effect of regular handling on fear reactions was investigated in mallard (Anas platyrhynchos) by exposing six hand-reared and four wild ducks to an approaching human being and recording heart rates with an external ECG device.
• 2.2. All ducks reacted to the approach with tachycardia, but the response was significantly less in tame birds.
• 3.3. Hand-reared females showed less response than males. No sex-linked differences were apparent in the wild ducks.
• 4.4. Decreasing responses throughout the experiments were only found in tame birds.
• 5.5. Fear or stress reactions can apparently be diminished through habituation induced by regular handling.

     

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.