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.

     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

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.

     

Distribution of lysosomal hydrolases in glomerular and tubular fractions of rabbit kidney cortex

• 1.1. The results on the distribution of lysosomal hydrolases indicated that the specific activity of acid phosphatase is 5 times higher in glomeruli compared to tubular fraction. The activity of gb-galactosidase was 4 times higher m tubules compared to glomeruli.
• 2.2. Sephadex G-150 gel chromatography of soluble fraction of cortex homogenate, glomeruli and tubules indicated that the enzyme acid phosphatase occurs in multiple forms designated as peaks I, II and III.
• 3.3. The specific activity of peak II was 12–15 times higher in glomeruli compared to the cortex homogenate, and very low in tubules.
• 4.4. Arylsulphatases A and B were also 2–3 times higher in the glomerular fraction compared to the tubules.

     

Purification and some properties of a Mg2+-activated acid phosphatase from rat testis

• 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
• 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
• 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
• 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
• 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Purification and properties of the proteinase inhibitors from Erythrina caffra (coast erythrina) seed

• 1.1. Four proteinase inhibitors (DE-1 to DE-4) were purified from Erythrinu caffra seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography involving DEAE-cellulose and DEAE-sepharose.
• 2.2. They comprise 164–166 amino acid residues (mol. wt 18,100) including 4 half-cystine residues and resemble the Kunitz-type proteinase inhibitors.
• 3.3. The N-terminal primary structure of DE-3 revealed also homology with those of the Kunitz-type inhibitors. For DE-1, DE-2 and DE-4 no free N-terminal amino acid was found.
• 4.4. DE-1 contains a potent inhibitor for both porcine trypsin and bovine α-chymotrypsin. Whereas DE-2 inhibits a-chymotrypsin strongly and has practically no action on trypsin, DE-3 inhibits both trypsin and a-chymotrypsin strongly. DE-4 is a potent inhibitor for trypsin but it binds a-chymotrypsin only weakly.

     

Purification and partial characterization of the alkaline phosphatase from Allolobophora caliginosa

• 1.1. A purification of the enzyme from the starting material was achieved by means of butanol and acetone fractionations and, successively, by DEAE cellulose and Sephadex G-200 chromatographies.
• 2.2. Two enzymatic forms were separated; they showed various similar characteristics but differed greatly in specific activity.
• 3.3. It is probable that in A. caliginosa a sole alkaline phosphatase form exists and the less active fraction is partly denatured enzyme.
• 4.4. It is not completely possible to exclude the existence of two isoenzymes.

     

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.

     

An enzyme-inhibitor complex of cathepsin L in the white muscle of chum salmon (Oncorhynchus keta) in spawning migration

• 1.1. A cathepsin L-inhibitor complex was purified from the white muscle of chum salmon (Oncorhynchus keta) by a series of chromatographies on DEAE-Sephadex, con A-Sepharose and Sephadex G-150.
• 2.2. The mol. wt of the complex was estimated to be 50,000 by gel filtration. The complex per se showed little activity of cathepsin L, but it became active when incubated at an acidic pH.
• 3.3. SDS-PAGE analysis and an experiment of activation by acidification indicated that the complex consisted of the 37 or 30 kDA-form of cathepsin L and the 15 kDa-endogenous cysteine protease inhibitor.
• 4.4. The enzyme-inhibitor complex was considered to be formed when cathepsin L leaks out of the lysosome in vivo or is freed from the lysosome when the tissue is artificially destroyed.

     

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.

     

Isolation and characterization of the major phospholipase a2 from the venom of trimeresurus purpureomaculatus (shore pit viper)

• 1.1. The major phospholipase A₂ (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity.
• 2.2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14.000 as estimated by SDS-polyacrylamide gel electrophoresis.
• 3.3. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC > PE > PS = 0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme.
• 4.4. The phospholipase A₂ exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 μg/g by i.v. route.

     

Partial purification and characterization of intestinal alkaline phosphatase of the rainbow lizard Agama agama

• 1.1. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase EC 3.1.3.1.) was extracted from the small intestines of the rainbow lizard Agama agama, partially purified by DEAE-cellulose and Sephadex G-200 column chromatography and characterized.
• 2.2. The enzyme had an optimum pH at 9.5 in sodium carbonate/bicarbonate buffer: a K_m of 1.6 mM with p-nitrophenyl phosphate; a molecular weight of 132,000; was inhibited by Zn²⁺, EDTA, urea and phenylalanine; stimulated by Co²⁺, Mn²⁺ and Mg²⁺, but Ca²⁺ had little or no effect on the activity of the enzyme.
• 3.3. The inhibition by urea was non-competitive, that by phenylalanine was uncompetitive. The enzyme was heat-labile.

     

Creatine kinase from the electric organ of Electrophorus electricus (L.)—isozyme analysis

• 1.1. Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract.
• 2.2. Upon acetone fractionation of the aqueous extract of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity.
• 3.3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure.
• 4.4. The implications of this finding with the muscular origin of the electric organ are discussed.

     

Isolation and partial characterization of a porcine low molecular weight protein occurring in plasma and urine

• 1.1. A low molecular weight (LMW) glycoprotein was isolated in the pig from urine produced after the induction of proximal tubular damage and uremia by maleic acid.
• 2.2. The purification steps included ultrafiltration, gel chromatography on Sephadex and anion exchange chromatography.
• 3.3. The molecular weight, determined by SDS-polyacrylamide electrophoresis was 12,500. The protein appeared heterogeneous in agarose gel electrophoresis. Immunoelectrophoresis and crossed immuno-electrophoresis demonstrated 2 major zones in the α-region, a minor in the early α₁- and one in the β-region.
• 4.4. Like the human LMW proteins it appeared in trace amounts in normal plasma and urine but its characteristics were unlike any of the known human plasma LMW proteins.

     

Isolation and N-terminal sequence of two low molecular weight calcium-binding proteins from pig granulocytes

• 1.1. Two small, abundant calcium-binding proteins were isolated from pig granulocytes. They were named p7A and p7B. Relative molecular masses were approx. 32,000 for p7A and 13,000 for p7B, when obtained by Sephadex G-75 gel filtration, while it was 7000 for both proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
• 2.2. N -terminal sequence analysis suggests that p7A is homologous to human and mouse MRP-8 and that p7B may be related to human and mouse MRP-14, though some properties of the latter—such as mobility on SDS-PAGE—were found to be different. In addition, p7A and p7B could be resolved under native conditions, contrasting with the fact that human and mouse MRP-8/MRP-14 form noncovalent complexes.

     

Properties of an alkaline phosphatase from the dinoflagellate Peridinium cinctum

• 1.1. Alkaline phosphatase (EC 3.1.3.1) from the dinoflagellate Peridinium cinctum, the Lake Kinneret bloom alga, has been partially purified by gel filtration.
• 2.2. The enzyme could be easily extracted using a distilled water/chloroform mixture suggesting that the alkaline phosphatase of Peridinium is particularly labile.
• 3.3. The molecular weight of the enzyme was estimated as 158,000 ± 5000. The enzyme showed a broad pH optimum (in the range pH 8.0–8.5), had a K_m of 0.45 mM for p-nitrophenylphosphate as substrate and was stable to repeated freeze/thawing cycles.
• 4.4. The enzyme was strongly activated by Mg²⁺ whereas Zn²⁺ (and to a lesser extent Cd²⁺) was an effective inhibitor of the enzyme. Cu²⁺ activated the enzyme at low concentrations, although at higher concentrations inhibited the enzyme. This effect of metals on the Peridinium alkaline phosphatase could be environmentally important since underwater hot springs, containing high concentrations of copper, enter the lake.

     

A novel pigment protein in the ovary of echinothurid sea urchins,Asthenosoma ijimai and Araeosoma owstoni

• 1.1. A protein conjugated with a pigment, which showed a peak of absorbance at 385 nm, was identified and partially purified from the ovary of Asthenosoma ijimai and Araeosoma owstoni by butanol extraction, gel filtration, ion-exchange chromatography and adsorption chromatography. This protein was observed only in ovaries, but not in testes.
• 2.2. This protein of A. ijimai showed a molecular weight of 600 kDa on gel filtration. The isoelectric point of the protein was 4.7.
• 3.3. The possible presence of this protein was examined by gel filtration chromatography in the ovaries of 11 other species of sea urchins, Glyptocidaris crenularis, Diadema setosum, Temnopleurus hardwicki, Toxopneustes pileolus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius, S. nudus, Echinostrephus aciculatus, Anthocidaris crassispina, Echinometra mathaei and Echinocardium cordatum. However, it was not detected.

     

Lysosomal acid deoxyribonuclease from vervet monkey livers—I: Purification and physico-chemical characterization

• 1.1. Acid DNase from monkey liver lysosomes was purified to homogeneity by salt extraction of lysosomal membranes at pH 3.8; (NH₄)₂SO₄ fractionation; low salt precipitation; SP-C₅₀ and G-150 Sephadex chromatography; and polyacrylamide gel electrophoresis.
• 2.2. The pH for optimum activity was dual in character with a labile optimum at pH 3.8 and a less active but stable one at pH 4.2
• 3.3. The estimated molecular weight was 40 K and the pl was 4.4.
• 4.4. Inorganic ions such as Ca²⁺, Mg²⁺, Mn²⁺ and SO₄²⁻ were more than 80% inhibitory at 10-mM levels. Fe³⁺ ions were 80% inhibitory at 0.1-mM levels. 15. Nad at 100 mM is essential for activity but becomes 100% inhibitory above 200 mM.

     

Purification,catalytic and regulatory properties of malate dehydrogenase from the foot of Patella caerulea (L.)

• 1.1. Malate dehydrogenase has been purified from the foot muscle of Patella caerulea by ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Blue Agarose and gel filtration on Sephadex G-150.
• 2.2. The yield was 23.5% of the initial activity with a final specific activity of 257 U/mg of protein.
• 3.3. The apparent mol. wt of the native enzyme is approx. 75,000 and it consists of two subunits of mol. wts in the range of 36,000–39,000.
• 4.4. The enzyme exhibits hyperbolic kinetics with respect to oxaloacetate, NADH and l-malate. The K_m values were determined to be 0.055 mM for oxaloacetate, 0.010 mM for NADH and 0.37 mM for l-malate. The pH optima are around 8.4 for the reduction of oxaloacetate and 9.2–9.6 for the reduction of oxaloacetate and 9.2–9.6 for the l-malate oxidation. V_max and K_m values for oxaloacetate change in an opposite manner with respect to pH values.
• 5.5. Of the various compounds tested, only α-ketoglutarate, citrate and adenylate phosphates were found to inhibit the enzyme activity.
• 6.6. From the above properties it appears that the reaction of cytoplasmic malate dehydrogenase of P. caerulea foot muscle is a key reaction in the anaerobic pathway and it occurs with the production of malate.