Comparative Biochemical Studies on α-Glucosidases

The substrate specificity of Schizosaccharomyces pombe α-glucosidase has been investigated.

The range of opt. pH was 4.0~4.4 and opt. temp. 45°C. The enzyme lost almost all the enzymatic activity at 55°C for 15 min. and the range of pH-stability was 3.2~6.8 at 30 °C for 20 hrs.

The enzyme was active especially on maltose in comparison with other α-glucosides. The rate of hydrolysis decreased in the following order: maltose, isomaltose, phenyl-α-glucoside, panose and turanose. Therefore, the substrate specificity of the enzyme was quite different from that of brewer’s yeast (Saccharomyces cerevisiae) α-glucosidase, though similar to that of mold α-glucosidase.     

Studies on α-Glucosidase in Rice

Two kinds of αglucosidase which were homogeneous in disc electrophoretic and ultra-centrifugal analysis were isolated from rice seeds by means of ammonium sulfate fractionation and CM-cellulose, Sephadex G–100 and DEAE-cellulose column chromatography and designated as α-glucosidase I and α-glucosidase II.

Both α-glucosidases hydrolyzed maltose and soluble starch to glucose and showed same optimal pH (4.0) on the both substrates. In addition, both enzymes acted on various α-linked gluco-oligosaccharides and soluble starch but little or not on α-linked hetero-glucosides and α-l,6-glucan (dextran).

Activity of the enzymes on maltose and soluble starch was inhibited by Tris and erythritol. α-Glucosidase II was more sensitive to the inhibitors than α-glucosidase I.

Km value for maltose was 1.1 mM for α-glucosidase I and 2.0 mM for α-glucosidase II.     

Biochemical Studies on Buckwheat α-Glucosidase

The transglucosylation action of buckwheat α-glucosidase on soluble starch, maltose maltotriose and maltotetraose are described and discussed. The transglucosylation products of soluble starch were isolated by carbon-Celite column chromatography and by paper chromatography. Among the products were found follows: nigerose, maltose, kojibiose and isomaltose as disaccharides and 2-α-isomaltosylglucose, 2-α-nigerosylglucose, nigerotriose. 3-α-maltosylglucose and maltotriose as trisaccharides.

Furthermore, the existence of 6-α-nigerosylglucose, 4-α-kojibiosylglucose, panose, isopanose and 3-α-isomaItosylglucose was suspected. A new trisaccharide, 2-α-nigerosylglucose which was obtained in a crystalline form (monohydrate) melted at 186~188°C and gave [α]_D+ 178.3 (c = 0.6, in water).

These experimental results on the reaction products seem to indicate that the activated glucosyl group from the substrate (starch, in this case) is transferred to any position of C–2,3,4 or 6 of glucose released from the substrate and the same type of transglucosylation occurrs upon the non-reducing terminal of disaccharides just produced, which leads to the formation of various kinds of trisaccharide, tetrasaccharide etc. The synthesis of α-oligosaccharides from free glucose could not be detected by paper chromatography.     

A Comparative Analysis of Interhelical Polar Interactions of Various α-Helix Packings in Proteins

One hundred twenty globular proteins and forty five leucine zippers representing all types of packing of long -helices were studied in terms of revealing and comparing their interhelical hydrogen and salt bonds. Many previous studies of leucine zippers and their analogs showed that interhelical interactions between polar groups could impart specificity to packing of an -helix. The current comparison demonstrated that basically, globular proteins and leucine zippers had similar interhelical polar interactions with presumably a similar structural role. However, depending on the packing of -helices, the networks of interhelical polar bonds were shown to be distinct and determined both by physicochemical properties of involved amino acid residues and by the relative positions of hydrophobic and hydrophilic residues on the surface of -helices. The revealed distinction is probably crucial for selecting the unique packing of an -helix.     

Studies on α-Alkyl-α-amino Acids

Effects of cytokinins were studied on rotenone-sensitive NADH dehydrogenase in mitochondria from fresh potato tubers (Solarium tuberosum), in consideration of the operation of external and rotenone-insensitive internal NADH dehydrogenases that has not been fully accounted for in previous studies. In submitochondrial particles (smp), zeatin was only weakly active, and zeatin riboside (ZR) was inactive. Inhibition rates at 400 μM of isopentenyladenine (iP) and isopentenyladenosine (iPA) were 45% and 30%, respectively, and that of BA (BA) was 64%. In intact mitochondria, the inhibition by iP and BA significantly increased, I₅₀ being 50 and 250 μM, respectively, but that by zeatin and iPA decreased. A structure–activity study showed that hydrophobic and steric factors are important for the activity. Cytokinins inhibited the electron flow via natural quinone more strongly than that via synthetic quinone. These results suggest that among the cytokinins the species that can regulate the electron transport is iP rather than its riboside or zeatin.     

Kinetic Studies on α-Aminoisobutyrate Decomposing Enzyme

An enzyme which catalyzes a decomposition of α-aminoisobutyrate (AIB) was purified and its kinetic properties were investigated. Michaelis constants for AIB decomposing reaction are able to be calculated by Ping Pong initial velocity equation. This enzyme catalyzes also l-alanine: α-ketobutyrate transamination as well as AIB decomposing reaction. Approximately equal values of Michaelis constants were obtained for α-ketobutyrate and pyridoxal 5′-phosphate (PLP), which are common substrates of both reactions.

In higher concentration of the enzyme, transamination between PLP and AIB or l-alanine was detected, whereas the reaction between pyridoxamine 5′-phosphate and pyruvate was not observed. These results are probably ascribed to a difference in affinity of two coenzymes for the enzyme.     

Studies on immobilized α-glucosidase from brewers yeastSaccharomyces carlsbergensis

Summary -Glucosidase isolated from brewer's yeast (Saccharomyces carlsbergensis) was immobilized using hydroxymethacrylate activated by cyanogen bromide as a carrier. Up to a hundred-fold increase in the stability of the enzyme was observed after immobilization. The yield in activity (bound/applied) was up to 30%. Before developing the process of enzymatic cleavage of maltose further we evaluated the kinetic properties of the enzyme catalyst, as we had observed earlier that the soluble enzyme is strongly inhibited by the product glucose. This is even more pronounced with the immobilized -glucosidase leading in this case to a linear relation between initial rate and substrate concentration, so K_M (approx.) values can no longer be defined due to the dominating influence of the product inhibition.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Critical Main-Chain Length for Conformational Conversion From 310-Helix to α-Helix in Polypeptides

Abstract

To assess the minimal peptide length required for the stabilization of the a-helix relative to the 3₁₀-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula -(Aib-L-Ala)_n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3₁₀-helical with four 1 ← 4 intramolecular N-H … O=C H-bonds. On the other hand, the octapeptide molecules are essentially α-helical with four 1 ← 5 H-bonds; however, the helix is elongated at the N-terminus, with two 1 ← 4 H-bonds, giving these molecules a mixed α/3₁₀-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3₁₀ →α-helix conversion in the crystal state induced by peptide backbone lengthening only.     

Enhancement of Stability of Double and Triple Stranded DNA by Cationic Amphiphilic α-Helix Peptide

Abstract

It was found that synthetic amphiphilic α-helix peptide could bind stabilize double or triple stranded DNA. The stabilization effect was significant for cationic α-helix peptides which indicated the importance of electrostatic interaction of positive charge of peptide and negative charge of DNA. It was also pointed out that α-helix content was increased in the presence of DNA.     

Physical Studies on DNA From “Primitive” Eucaryote

Abstract

INTRODUCTION

Studies carried out over the years on bacteria and higher eucaryotes show several major differences between the two types of organisms in the nature of the DNA and chromosomes. Some of the differences are

1. The existence of a large percentage of interspersed, repetitive sequences in higher eucaryotes but not in bacteria.

2. The existence of chromatin containing large amounts of basic (histone) and nonhistone chromosomal proteins in higher eucaryotes but not in bacteria.

3. Bidirectional DNA replication from many initiation sites found in higher eucaryotes as compared to the single initiation site found in bacteria.     

Chemical Studies on Amino—Carbonyl Reaction

When N-n-butyl-D-xylosylamine was heated with acetic acid in methanol at 55~70°G, it decomposed to N-n-butyIpyrrole-2-aldehyde,** through 3-deoxy-d-pentosulose as an intermediate. d-Xylose and methylamine in neutralized aqueous solution at 65~100°C also formed N-methylpyrrole-2-aldehyde. N-n-Butyl-l-rhamnosylamine, in a mixture of methanol and acetic acid, formed the corresponding pyrrolealdehyde, l-n-butyl-5-methylpyrrole-2- aldehyde, at the almost same rate as did N-xyloside. On the contrary, N-n-butyl-d-glucosylamine, under the same condition, did not form any detectable amount of the corresponding pyrrolealdehyde, but formed complicated products. A formation mechanism of the pyrrolealdehydes from 3-deoxyosulose and amine was proposed.     

Studies on the thermostability of the α-amylase of Bacillus caldovelox

Summary Molecular mechanisms of thermoinactivation of the thermostable -amylase of Bacillus caldovelox were examined. Monomolecular conformational processes were found to be the major causes of thermoinactivation at both pH 4.5 and 8.0. The enzyme possessed considerable additional thermostability at pH 8.0, with half-lives of 0.75 and 7.0 min at 90° C and pH 4.5 and 8.0, respectively. The amino acid composition was examined with respect to the underlying thermostability exhibited by this enzyme. The inherent thermostability exhibited may be due to the high proline content (4.47 mol%), but more likely due to the high content of residues forming hydrophobic bonds (60.89 mol%) allied to a low content of residues responsible for ionic interactions (28.34 mol%). Offprint requests to: C. T. Kelly     

Studies on α-D-mannosidase and ConA during jackbean development and germination

Summary Changes in the level and activity of -D-mannosidase and ConA have been followed during a time-course of development spanning seed formation, desiccation and germination. In parallel studies, immunogold labelling has enabled these changes to be placed within a structural context of the cotyledon parenchyma cells during protein body formation, dehydration and subsequent autolysis during germination.The results indicate that the exo-glycosidase and lectin accumulate in parallel during seed formation and are packaged within the same protein bodies. Several lines of evidence suggest that the function of both proteins is related to events that occur during seed development rather than germination.Abbreviations BSA bovine serum albumin - ConA concanavalin A - DPA days post-anthesis - DPI days post-imbibition - Man D-mannose - PBS phosphate buffered saline - RIA radioimmunoassay - ER endoplasmic reticulum - GA Golgi apparatus - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate     

Studies on the Decomposition of Raffinose by α-Galactosidase of Mold

A mold which produced α-galactosidase and little invertase was isolated and identified as Mortierella vinacea. α-Galactosidase formation of the mold was induced by galactose, melibiose, raffinose and lactose. Among these inducers lactose showed the most stimulative effect. α-Galactosidase was produced by either Koji method or submerged culture method, but in the latter most α-galactosidase was found in the mycelium fraction.

Hydrolysis of raffinose in beet molasses was studied with the α-galactosidase in the mycelium fraction and about 80% of raffinose was found to be hydrolyzed by the enzyme preparation.     

Studies on the Decomposition of Raffinose by α-Galactosidase of Actinomycetes

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl₂ and AgNO₃ caused complete inhibition of the enzyme activity at 2 × 10^?5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.

A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.     

Critical Main-Chain Length for Conformational Conversion From 3(10)-Helix to α-Helix in Polypeptides

To assess the minimal peptide length required for the stabilization of the alpha-helix relative to the 3(10)-helix in Aib-rich peptides, we have solved the X-ray diffraction structures of the terminally blocked sequential hexa- and octapeptides with the general formula-(Aib-L-Ala)n-(n = 3 and 4, respectively). The hexapeptide molecules are completely 3(10)-helical with four 1----4 intramolecular N-H . . . O = C H-bonds. On the other hand, the octapeptide molecules are essentially alpha-helical with four 1----5 H-bonds; however, the helix is elongated at the N-terminus, with two 1----4 H-bonds, giving these molecules a mixed alpha/3(10)-helical character. In both compounds the right-handed screw sense of the helix is dictated by the presence of the Ala residues of L-configuration. This study represents the first experimental proof for a 3(10)----alpha-helix conversion in the crystal state induced by peptide backbone lengthening only.     

Studies on type II glycogenosis. Effects of cortisone derivatives on acid α-glucosidase

Cortisone causes a marked increase in the activity of liver acid alpha-glucosidase 2h after injection into male Wistar rats. Studies on rat liver tissue slices, isolated lysosomes and cultured skin fibroblasts have demonstrated similar elevations of acid alpha-glucosidase activity after incubation with cortisone. Cortisone-treated human liver tissue, obtained by needle biopsy, also shows an increase in acid alpha-glucosidase activity. Neutral alpha-glucosidase activity was not stimulated by cortisone in vivo or in liver slices.