Takehiko Yamamoto

A novel method of isolation and some characteristic properties of human pancreatic elastases

One component of elastases of human pancreatic juice and pancreatic extract was obtained in a highly purified state by chromatography on a column of sawdust. The elastase obtained after repeated adsorption chromatography with NaCl-containing buffers was almost homogeneous by gel filtration and polyacrylamide gel electrophoresis. This elastase showed relatively high elastolytic activity, but relatively low hydrolytic activity towards succinyl trialanine p-nitroanilide, as compared with another component of pancreatic juice elastase (which was not absorbed onto sawdust). Both elastases isolated were alkaline earth metal-dependent enzymes.

Development of a radioimmunoassay for human deoxyribonuclease I

A reliable radioimmunoassay (RIA) for human deoxyribonuclease I (DNase I) is described. Using delayed addition of tracer antigen, the method is sensitive (9.5 reproducible and specific. A good parallel relationship was observed between the standard curve and dilution curves for human urine and human pancreatic juice. G-actin, a naturally occurring DNase I inhibitor, caused no change in the immunoreactivity of DNase I. In healthy individuals, aged 11-90 yr, the mean serum DNase I was 18.4 ng/ml (SD 6.7 ng/ml). Increased serum DNase I occurred in patients with acute pancreatitis, renal failure, and in about one-third of patients with various malignant tumors.

Modification of human pancreatic amylase isozymes by peptidoglutaminase I and II.

The effects of peptidoglutaminase (PGln-ase) I and II on human pancreatic juice amylase purified as an isozyme were investigated. Several amylase isozymes were formed which corresponded to minor components of pancreatic amylase isozymes, indicating that appearance of amylase isozymes are due to enzymic deamidation. A similar result was observed when the purified amylase isozyme was incubated with the supernatant of human pancreatic juice whose amylase was previously removed by adsorption onto raw corn starch. These findings are discussed in connection with amylase isozymes in the sera of the patients suffering from pancreatic inflammation.

Studies of Hemicellulolytic Enzymes of Bacillus subtilis :Part I. Purification, Crystallization and Some Properties of Arabinogalactanase

Many strains of Bacillus subtilis were found to secrete several hemicellulolytic enzymes such as arabinoxylanase, galactomannanase, arabinogalactanase, etc. Chemical and enzymatic properties of certain strains of the bacterium were comparatively investigated. An arabinogalactanase was purified and obtained in a crystalline state. Its molecular weight and isoelectric point were estimated to be 3.7×104 and 8.39, respectively. The enzyme showed an optimal pH for reactions at 6.0, and was stable in a pH range of 5.0 to 9.5 at 30°C. It required no metallic ions for its activity and hydrolyzed soybean arabinogalactan, forming galactobiose as the main product. No liberation of arabinose was observed in the hydrolysate. Also, the enzyme did not attack coffee bean arabinogalactan. Some implications of the experimental results are discussed.

Arabinogalactanase of Bacillus subtilis var. amylosacchariticus

Publisher Summary Many strains of Bacillus subtilis and Bacillus amyloliquefaciens are good producers of several enzymes and some of them or their mutant strains have been industrially utilized for production of α-amylase and proteinase. These bacteria are also a good source of the enzymes that hydrolyze “hemicelluloses” of plant cell wall such as arabinogalactan, galactomannan , and arabinoxylan . This chapter discusses the method of purification and crystallization of arabinogalactanase, starting from the cultured liquor of a strain of saccharifying a-amylase producing Bacillus subtilis var. amylosacchariticus , and showing the separation stages of α-amylase and proteinase that are simultaneously produced in the cultured liquor. The chapter describes the properties of the enzyme indicating that it is a metal ion requiring enzyme for the stability and that it hydrolyzes endwise the arabinogalactan of soybean seed to produce galactose and several galactooligosaccharides with or without arabinose residues. The chapter discusses the properties of arabinogalactanase obtained from another bacterial strain.

Studies on Invertase of Candida utilis Part I:A Comparative Study on Isolation Methods of the Enzyme

Invertase (β-d-fructofuranoside fructohydrolase, 3.2.1.26) of Candida utilis was obtained not only from the autolysate of the cells, but also from the culture filtrate and the supernatant of the aerobically incubated cell suspensions. The invertase formed in the yeast cells was not influenced by the kind of sugar used as a carbon source for culture, and the release of the enzyme from the cells occurred under aerobic condition. The addition of sugar to the aerobically incubated cell suspension prevented the enzyme release. Liberation of invertase from the yeast cells was also found to occur under another condition, and the procedure designated as the salt-steeping method was described in the present paper.Among the invertase preparations by the above methods, however, the preparation from the aerobically incubated cell suspension was most pure and obtained easily in a highly purified state by treating with columns of DEAE-cellulose and Sephadex G-200.

Proteochemical, immunological and enzymatic properties of two amylase components from human pancreatic juice.

Two amylase components were purified from human pancreatic juice and investigated for their proteochemical, immunological and enzymatic properties. The two components were similar in molecular weight, amino acid composition and immunoreactivity as determined by radioimmunoassay for the major component of pancreatic amylase. Their specific activity, optimal pH value, and stability were identical. Both were equally activated by chloride ion and their hydrolysis modes of various substrates were also identical. The only difference found between them was in the electrophoretic mobility in the peptide map, indicating that the difference was brought about by deamidation. The quantitative analysis of pancreatic amylase isozymes can provide information on the posttranslational modification in pancreatic tissue and/or in body fluids.

Bitter Peptides in Cow Milk Casein Digests with Bacterial Proteinase: Part I. Isolation and Determination of Amino Acid Sequence of a Bitter Peptide

Isolation and determination of amino acid sequence of the bitter peptides formed in the digestion of cow milk casein with alkaline proteinase of Bacillus subtilis were investigated. The casein digest with the enzyme was extracted with butanol and the extracted bitter peptides were fractionally purified by treating with several other organic solvents followed by subjecting to chromatography and gel-filtration. The amino acid sequence of one of the bitter peptides was determined as follows: Arg-Gly-Pro-Pro-Phe-Ileu-Val. Liberation of N-terminal Arg with trypsin or bacterial aminopeptidase did not affect the bitterness. Also, splitting off of Val and Ileu or Ileu-Val at the C-terminus by carboxypeptidase, or a bacterial neutral proteinase gave no influence on the bitterness. However, liberation of Arg and Gly from the peptide with bacterial aminopeptidase gave rise to a non bitter peptide.

Intracellular Peptidase of Bacillus subtilis :Part III. Effects of Metal Ions on Activity and Specificity of Aminopeptidase of Bacillus subtilis

An electrophoretically pure preparation of aminopeptidase was isolated from the cells of a strain of Bacillus subtilis which secreted saccharifying α-amylase. The purified peptidase was active only in the presence of manganese and cobaltous ions. Both the metallic ions were effective to a similar degree for the enzyme on most of the peptides used as the substrate. However, for hydrolysis of leucine amide by the enzyme was effective only cobaltous ion and, in this case, manganese ion showed to act as a competitive inhibitor. On the other hand, for hydrolysis of certain other peptides such as glycyl proline, quite the opposite relationship was observed between those metallic ions. In the present paper is also described a new micro-assay method of amidase activity shown by the peptidase.

A Glucomannan as an Extracellular Product of Candida utilis I. Production and Characterization of a GlucomannanII. Structure of a Glucomannan: Characterization of Oligosaccharides Obtained by Partial Hydrolysis

A wild yeast, Candida utilis ATCC 42402, was found to secrete a glucomannan together with invertase on the consumption of sugar from the medium. The glucomannan in the culture filtrate was readily separated from the invertase by DEAE-cellulose chromatography. The crude glucomannan thus obtained was fractionated on a column of DEAE-Sephadex A-25 into several types of molecular species. The fractions were characterized by their sugar and amino acid composition which showed a tendency for an increasing mannose/glucose ratio, (1.66-5.74) coincident with greater amounts of the proteinous moiety (0.6-6%). One fraction from the DEAE-Sephadex A-25 separation, which was distinct from the other fractions in its lack of glucosamine as a component, showed a high homogeneity (M.W., 7.4 × 104 Sw,20 = 6.11 S).The structure of an extracellular glucomannan produced by Candida utilis was studied. Up to 15% hydrolysis of the glucomannan with the α-mannosidase isolated from Arthrobacter GJM-1 did not alter its viscosity. Seq...