Kazuo Yonaha

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.

Purification of an antihemorrhagic factor from the serum of the non-venomous snake Dinodon semicarinatus

An antihemorrhagic factor was purified from the serum of Dinodon semicarinatus, a non-venomous snake (Akamata), by a series of high performance liquid chromatographies with a TSK gel DEAE-5PW column. The purified antihemorrhagic factor showed a single band on polyacrylamide gel disc electrophoresis and inhibited the hemorrhagic activity of HR1 and HR2, the hemorrhagic factors of Trimeresurus flavoviridis Okinawa. The antihemorrhagic factor was stable from 0 degrees to 60 degrees and at pH values 2.0-11.0. The molecular weight of the factor was estimated to be 59,000 and 52,000 by a gel filtration and SDS-disc electrophoresis, respectively, suggesting that it consists of a single subunit, as we also found for the antihemorrhagic factors of the mongoose Herpestes edwardsii.

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.

Neutralization of hemorrhagic snake venoms by sera of Trimeresurus flavoviridis (Habu), Herpestes edwardsii (mongoose) and Dinodon semicarinatus (Akamata)

The sera of T. flavoviridis (Habu), H. edwardsii (mongoose) and D. semicarinatus (Akamata, non-venomous snake) were tested for their capacity to neutralize 28 species of hemorrhagic snake venoms in vitro. The sera of these animals neutralized a variety of hemorrhagic venoms, suggesting a common structure for a hemorrhagic factor and a similar mechanism of neutralization for the antihemorrhagic factor in the sera. The serum of T. flavoviridis neutralized the lethal toxicity of the T. flavoviridis venom but could not neutralize those of the other hemorrhagic venoms at all. The sera of H. edwardsii and D. semicarinatus did not inhibit the activity of all hemorrhagic venoms.

Characterization of three hemorrhagic factors from the venom of Okinawa habu (Trimeresurus flavoviridis)

Three hemorrhagic factors, HR1, HR2a and HR2b, of Okinawa habu venom were characterized in terms of their subunit structure, amino acid composition, metal content and immunological properties. HR1 is a dimer (mol. wt 90,000) consisting of two identical subunits at 25 degrees C, but polymerizes to form a tetramer at 4 degrees C. Two peaks corresponding to the dimer and the tetramer were observed upon ultracentrifugation analysis at 20 degrees C. HR2a and HR2b are monomers (mol. wt 24,000 and 19,000, respectively). HR1, HR2a and HR2b contain 407, 203 and 161 amino acids, respectively and the respective mol. wt based on the amino acid composition are 45,988, 23,075 and 18,457. The hemorrhagic factors contain Zn2+, Ca2+ and Mg2+, and were irreversibly inhibited by incubation with chelating reagents. The three hemorrhagic factors were immunologically distinguished from each other, and the hemorrhagic activities were inhibited by the respective antiserum. The activity of HR2a was also inhibited by the antiserum against HR2b.

The primary structure of a hemorrhagic factor, HR2b, from the venom of Okinawa habu ({iTrimeresurus flavoviridis})

The complete amino acid sequence of a hemorrhagic factor, HR2b, from the venom of Okinawa habu was determined. The hemorrhagic factor was fragmented by CNBr cleavage, trypsin, staphylococcal protease V8 and lysyl endopeptidase digestions. The resulting peptides were purified on high performance chromatography, and sequenced by Edman degradation. HR2b was composed of 204 amino acids with pyroglutamyl residue at the amino terminus, and the calculated mol. wt based on the amino acid composition was 23,335. There are three disulfide linkages in the primary structure. The consensus sequence (His-Glu-Xaa-Xaa-His) for zinc-binding site of zinc-requiring metalloproteinases was found in the structure. The primary structure of HR2b shows a significant similarity with that of HR2a of Amami habu venom; 98.5% identity.

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.