Seizen Toyama

Purification of three antihemorrhagic factors from the serum of a mongoose (Herpestes edwardsii)

Three antihemorrhagic factors (AHF-1, AHF-2 and AHF-3) were purified from the serum of H. edwardsii, a mongoose, by a combination of gel filtration on a Sephadex G-200 column and high performance liquid chromatography with a TSK gel DEAE-5PW column. Each of the purified antihemorrhagic factors showed a single band on polyacrylamide gel disc electrophoresis. The three antihemorrhagic factors inhibited the hemorrhagic activity of HR 1 and HR 2, the hemorrhagic principles from the snake venom of Trimeresurus flavoviridis Okinawa. AHF-1, AHF-2 and AHF-3 were stable at temperatures from 0 degrees to 60 degrees C and at pH values between 2.0 and 11.0. The same molecular weight (65,000) was obtained for the three antihemorrhagic factors. No precipitin lines were found for the purified antihemorrhagic factors with the venom of T. flavoviridis Okinawa and its hemorrhagic principles, HR 1 and HR 2.

γ-Aminobutyrate:α-Ketoglutarate aminotransferase from Pseudomonas sp. F-126: Purification, crystallization, and enzymologic properties

Abstract γ-Aminobutyrate:α-ketoglutarate aminotransferase was purified from Pseudomonas sp. F-126 and crystallized. The crystalline enzyme is homogenous by the criteria of disc electrophoresis and ultracentrifugation ( s 0 20,w = 8.8 S). Molecular weights of 176,000 and 178,000 were obtained by gel filtration and ultracentrifugal sedimentation equilibrium methods, respectively. The enzyme exhibits absorption maxima at 415, 278, and 258 nm with shoulders at 284, 270, and 265 nm. The enzyme catalyzes the transamination of various ω-amino acid with α-ketoglutarate; γ-aminobutyrate is the best amino donor. The Michaelis constants are 2.8 m m for α-ketoglutarate and 4.1 m m for γ-aminobutyrate. In addition to ω-amino acids both isomers of ornithine and lysine and their decarboxylated product; i.e. , putrescine and cadaverine, can serve as amino donors. No activity was observed with β-alanine. The enzyme has a maximum activity in the pH range of 8.5–9.0 and at 60 °C. The enzyme is stable at pH 6.0–10.0 at temperatures up to 65 °C. Pyridoxal 5′-phosphate protects the enzyme from heat inactivation. Carbonyl reagents and sulfhydryl reagents inhibit the enzyme activity but the enzyme inactivated by these reagents was reactivated by incubation with pyridoxal 5′-phosphate and thiol compounds, respectively. Chelating agents, nonsubstrate l - and d -α-amino acids, and metal ions except cupric ion showed no effect on the enzyme activity.

Synergistic interaction between κ-carrageenan isolated from Hypnea charoides LAMOUROUX and galactomannan on its gelation

Abstract The synergistic effects on rheological properties for a series of aqueous solution of κ -carrageenan isolated from Hypnea charoides L amouroux and galactomannan (locust-bean gum) were investigated. At a concentration of 0.4% of total gums, gelation did not occur at room temperature, but it did at a low temperature (0°C). The maximum dynamic modulus was obtained with a series of the samples composed of K-salt of κ -carrageenan and locust-bean gum in the mixing ratio of 1:1 at low temperature (0°C). The less synergistic effect on the dynamic modulus was obtained in mixture solutions with Na-salt of κ -carrageenan and locust-bean gum. At about 25°C, gel-sol transition was observed in the mixing ratio of κ -carrageenan (K-salt) to locust-bean gum of 3:1 and 4:1. A possible association site between K-salt of κ -carrageenan and locust-bean gum was proposed.

Characterization of the antihemorrhagic factors of mongoose (Herpestes edwardsii).

Three antihemorrhagic factors (AHF1, AHF2 and AHF3) isolated from the serum of mongoose (Herpestes edwardsii) are glycoproteins of monomer structure with the same mol. wt (about 65,000), which contain 4.2%, 13.6% and 6.0% carbohydrates as glucose, respectively. All are composed of about 600 amino acids of similar composition. The 32 amino terminal amino acid sequences of three antihemorrhagic factors were determined, and sequence homologies were examined. AHF1 and AHF2 were of the same amino acid sequence, and showed high homologies to AHF3, oprin (opossum proteinase inhibitor) and human alpha 1B-glycoprotein; 68.7%, 42.3% and 50.0% identity, respectively. AHF1 completely inhibited the hemorrhagic activity of HR2b, the hemorrhagic factor of habu snake, at the concentration of five-fold molar excess, although incomplete inhibition (50%) of proteinase activity of the hemorrhagic factor was observed even at the concentration of 20-fold molar excess of antihemorrhagic factor. Incubation of HR2b with AHF1, and analysis of the reaction products by chromatography on TSK gel G-3000SW and on the ultracentrifuge did not show formation of an inactive enzyme inhibitor complex. However, the complex formation between AHF1 and HR2b was observed by a BIAcore analysis and TSK gel SP-5PW column chromatography. No alteration in the primary or the secondary structure of both factors was demonstrated by SDS-PAGE and circular dichroism spectrum at the far-UV wavelength before and after incubation of both factors, respectively.

Isolation of peptides homologous to domains of human α 1B-glycoprotein from a mongoose antihemorrhagic factor

Thirteen peptides, homologous to one of the five domains of human alpha 1B-glycoprotein (alpha 1BG), were isolated from a mongoose antihemorrhagic factor (AHF1); four of them were generated by BrCN cleavage, and three and six peptides by the digestions with lysyl endopeptidase and staphylococcal protease V8, respectively. The purified peptides covered 75.9% of the whole sequence of human alpha 1BG (359/474 residues) and showed 46.4% identity (167/360 amino acids) with the sequence of human alpha 1BG including cysteine residues forming disulfide linkages. One of the sugar binding sites of human alpha 1BG was also conserved in AHF1. These results suggest that AHF1 is a protein homologous to human alpha 1BG and a supergene family of immunoglobulins.

Purification and some properties of a thermostable xylanase from thermophilic fungus strain HG-1

Abstract An extracellular xylanase was purified to homogeneity from a wheat bran culture of the thermophilic fungus HG-1, an isolate from a compost heap. The enzyme had a molecular weight of 33,000 by SDS-PAGE and 31,000 by gel filtration; its isoelectric point was 6.8. The optimum temperature and pH for enzyme activity were 70°C and 4.5–5.0. The enzyme was stable in the pH range from 2 to 12 at 30°C. The K m values for birchwood xylan and oat-spelt xylan were 8.3 and 20 mg/ml, respectively. The enzyme produced xylobiose, xylotriose, and a trace of xylose as the endo products from birchwood xylan. The enzyme activity was strongly inhibited by SDS, and partially by Hg 2+ , Mn 2+ , Co 2+ , Ca 2+ , and iodoacetic acid. The enzyme hydrolyzed xylotriose to xylobiose and xylose and showed weak activity toward xylobiose.

Disintegration of Uncooked Rice by Carboxymethyl Cellulase from Sporotrichum sp.HG-I

A thermostable carboxymethyl cellulase (CMCase) was purified to homogeneity from a wheat bran culture of the thermophilic fungus Sporotrichum sp. HG-1, an isolate from a compost heap. The enzyme had a molecular weight (M(r)) of 33,000 by SDS-PAGE. The optimum temperature and pH for enzyme activity were 70 degrees C and 4.5-5.0, and the enzyme was heat stable. Uncooked Thai rice was digested so as to cause its disintegration by the addition of purified CMCase, but not by the addition of xylanase purified from strain HG-1. Ferulic acid conjugated to oligosaccharide was released significantly by the combined action of CMCase and xylanase, but the free form of ferulic acid was not detectable.

Purification and crystallization of hemorrhagic factor, HR2b, from the venom of Trimeresurus flavoviridis (habu)

The hemorrhagic factor, HR2b, was purified from the venom of Trimeresurus flavoviridis (habu) by a combination of gel filtration, cation exchange column chromatography and high performance liquid chromatography. The purified HR2b was homogeneous by the criteria of ultracentrifugation and SDS-disc electrophoresis. The mol. wt of HR2b was 18,000 and 18,500 by gel filtration on Sephadex G-50 and by SDS-disc electrophoresis, respectively, indicating a monomer structure for the hemorrhagic factor. Crystals of HR2b, taking the form of thin plates, were obtained in the presence of ammonium sulfate.

Enzymatic determination of l-alanine with ω-amino acid:pyruvate aminotransferase

Abstract A simple and specific method with bacterial ω-amino acid:pyruvate aminotransferase and lactate dehydrogenase has been reported for the determination of l -alanine. This method involves a transamination of l -alanine with sulfoacetaldehyde to produce pyruvate and the spectrometric determination of this product with the aid of lactate dehydrogenase.