Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

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.

     

Induction of a cadmium-binding protein in a unicellular alga

• 1.1. An inducible Cd-binding compound has been detected in a Cd-treated euryhaline alga, Dunaliella bioculata.
• 2.2. The apparent molecular mass of this heat-stable Cd-binding compound, as determined by gel filtration, was about 10,000.
• 3.3. Anion exchange chromatography (on DEAE Sepharose and Mono Q) and autoradiography of non-denaturing PAGE of the M_r 10,000 fractions revealed the presence of a highly acidic Cd-binding protein which differs on its properties from mammal metallothioneins.

     

Purification and characterization of a blue-colored red-fluorescent protein from the silkworm (Bombyx mori L.) larvae

• 1.1. A red-fluorescent blue protein (P600) was purified from the digestive juice of the silkworm (Bombyx mori L.) larvae raised on mulberry leaves.
• 2.2. The purified protein was electrophoretically homogeneous and showed the absorption maxima at 601.5 nm and 278 nm, and the fluorescence maximum at 621 nm.
• 3.3. The molecular weight was estimated to be 540,000 by gel filtration on Sepharose CL-6B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the protein consists of two heterogeneous polypeptide subunits with a mol. wt of 15,000 and 18,000.
• 4.4. The P600 contains excess acidic amino acid residues over basic groups. The polarity and pI were 45.5% and 4.6, respectively.
• 5.5. The production of H₂O₂ was observed in the presence of P600 upon illumination.

     

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.

     

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.

     

Two acid phosphatases in sea urchin eggs and embryos

• 1.1. Two types of acid phosphatases from sea urchin eggs and embryos were studied in three Japanese species, Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus.
• 2.2. Acid phosphatase type 1, designated AcP-1, hydrolysed only flavin mononucleotide besides p-nitrophenylphosphate. The activity of AcP-1 was not inhibited by NaF and tartrate. This enzyme showed molecular weight between 14,000 and 16,000 by gel filtration through Sephadex G-75.
• 3.3. The higher molecular weight type of acid phosphatase, designated AcP-2, showed relatively high substrate specificity toward ADP and ATP. Molecular weight of AcP-2 ranged from 42,000 to 48,000 by gel filtration through Sephadex G-100.
• 4.4. Some properties of AcP-2 from Sphaerechinus granularis embryos are also described.

     

Copurification and separation of β-galactosidase and sialidase from porcine testis

• 1.1. A soluble sialidase was copurified apparently as an enzyme complex with acid β-galactosidase from porcine testis.
• 2.2. The sialidase exhibited its maximum activity at acidic pH. It was efficiently active towards 4-methylumbelliferyl-α-d-N-acetyl-neuraminic acid and sialyllactose, relatively inactive towards glycoproteins, and had little activity towards glycolipids.
• 3.3. The complex could be separated by sucrose gradient centrifugation or isoelectric focusing.
• 4.4. The separated enzymes had molecular weights about 600,000 for β-galactosidase and more than about 1,000,000 for sialidase by Sepharose 4B gel filtration.
• 5.5. SDS-polyacrylamide gel electrophoresis of the β-galactosidase showed three protein bands with molecular weights of 63,000, 31,000 and 20,000.

     

Purification,characterization and kinetic mechanism of glucose-6-phosphate dehydrogenase from mouse liver

• 1.1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE.
• 2.2. The native and subunit molecular weight were 117 and 31 kDa respectively.
• 3.3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism.
• 4.4. The reduced K_m values for the substrates favour the operativity of the enzyme. The “fine control” of the enzymatic activity was exerted by the NADPH, whose K_i is several fold lower than the in vivo concentration.

     

The major dog pancreas protein recognized by an antiserum to dog prostate kallikrein is the anionic trypsin

• 1.1. On the basis of its immunoreactivity with a polyclonal antiserum to dog prostate kallikrein in Western blot experiments, a 30 kDa protein was purified from the pancreas of the dog using ion-exchange and gel filtration chromatography.
• 2.2. That protein was identified as the anionic trypsin by its NH₂-terminal amino acid sequence.
• 3.3. The immunoreaction occurred despite an overall amino acid homology which was limited to 39% between the prostate kallikrein and anionic trypsin.
• 4.4. Otherwise, the anti-prostatic kallikrein antiserum was rather specific since it did not react with dog cationic trypsin, dog renal kallikrein and human prostate specific antigen.

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A comparative study of clothing factors from the venom of Agkistrodon acutus collected in China and Taiwan

• 1.1. Two clotting factors, Cf-1(C) and Cf-2(C) were isolated from Agkistrodon acutus (collected in China) venom by gel filtration, ion-exchange chromatography and affinity chromatography. Using the same procedure, two clotting factors, Cf-1(T) and Cf-2(T), were isolated from Agkistrodon acutus (collected in Taiwan) venom and their characteristics were compared with Cf-1(C) and Cf-2(C).
• 2.2. Molecular weights of Cf-1(C), Cf-2(C), Cf-1(T) and Cf-2(T) were determined to be 44,000, 70,000, 25,000 and 44,000 respectively. The factors were not immunologically related.
• 3.3. The four clotting factors possessed tosyl-l-arginine methyl ester (TAME) hydrolyzing activity and coagulated fibrinogen to fibrin. Only Aα chain was cleaved when fibrinogen was incubated with each factor.
• 4.4. Agkistrodon acutus is not classified by geographical location, however it is obvious that venom components vary between the Chinese and Taiwanese forms.

     

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.

     

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.

     

Protein characterization of sexually mature and immature forms of Schistosoma mansoni

• 1.1. Protein composition of different stages of Schistosoma mansoni was compared using specific antisera, 2D polyacrylamide gel electrophoresis and 14-C-leucine incorporation into proteins.
• 2.2. Major qualitative differences were detected when an anti-membrane antiserum was used.
• 3.3. 2D gel electrophoresis showed that the protein composition varied when mature and immature females were compared, whereas no differences were noted when mature and immature male worms were compared.
• 4.4. Experiments measuring protein synthesis by the different schistosome stages confirmed that upon maturation, only the female schistosomes displayed qualitative differences.
• 5.5. The protein pattern of the male schistosomes did not vary significantly as a function of development.

     

Evidence of presence of a low molecular weight,non-metallothionein-like metal-binding protein in the marine gastropod Nassarius reticulatus L.

• 1.1. A low molecular weight metal-binding protein was found in the snail Nassarius reticulatus cytosol, which was induced in heavy metal contaminated environments.
• 2.2. In our sodium dodecyl sulfate-mercaptoethanol polyacrylamide gel systems it behaved as a protein of 19 kDa mol. wt.
• 3.3. Amino acid composition studies definitely established this protein not to be metallothionein (Mt) like, because it had a much lower level of cysteine and substantial amounts of aromatic amino acids and histidine.
• 4.4. The metal-binding strength of this protein was concluded to be much weaker than that of Mt.
• 5.5. In the crustacean Pagurus bernhardus L. such a protein could not be demonstrated.
• 6.6. In both the snail and the crustacean Zn may inhibit the accumulation of Hg. The premise for studying the induction of the metal-binding Nassarius protein as a supplement to environmental metal monitoring purposes is briefly discussed.

     

Purification of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Amino acid composition and the protein form of procarboxypeptidase a purified from the pancreas of the sei whale Balaenoptera bolealis

• 1.1. Procarboxypeptidase (W-PCPA) was purified from the pancreas of the sei whale Balaenoptera bolealis.
• 2.2. W-PCPA was obtained as a homogeneous protein in polyacylamide gel disc electrophoresis.
• 3.3. W-PCPA has a molecular weight of 75,000.
• 4.4. Amino acid composition of W-PCPA was compared with that of bovine procarboxypeptidase as A S5 (PCPA-S5).
• 5.5. W-PCPA may be two subunits, and the aggregate form may resemble PCPA-S5.

     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.