Probes for Catalytic Action of α-Chymotrypsin in Plastein Synthesis

A plastein was synthesized with α-chymotrypsin from a dialyzable fraction of a peptic hydrolysate of soybean protein.

The plastein was obtainable also by use of an insoluble preparation of α-chymotrypsin. This may rule out the possibility that the plastein is a product resulting from some chemical peptide-protein (enzyme) aggregation.

No appreciable amount of the plastein was produced when chymotrypsinogen was used instead of α-chymotrypsin.

The plastein synthetic, as well as the protein hydrolytic, activity of α-chymotrypsin was inhibited more or less by a hydrophobic inhibitor (n-hexane), a competitive inhibitor (benzolyl-d,l-phenylalanine), and divalent cations (Zn²⁺, Hg²⁺ and Cu²⁺); the degree of inhibition in each case was approximately similar against both the synthetic and the hydrolytic activities.

Either diisopropylphosphorylation of the β-O of Ser-195 or methylation of the 3-N of His-57 imidazole of α-chymotrypsin repressed the synthetic, as well as the hydrolytic, activity.

Based on these results a possible mechanism was discussed of the plastein synthesis by α-chymotrypsin, especially in relevance to its acylation and deacylation.     

Isolation and Identification of a Hog Pancreatic α-Amylase Inhibitor (Haim) Producing Streptomyces

A screening test was carried out to obtain microbes which produce hog pancreatic α-amylase inhibitor and a new inhibitor was found in culture broth of an actinomycete, strain YM-25. This inhibitor was designated as Haim, an abbreviation for hog pancreatic α-amylase inhibitor from a microbe. The determined morphological and physiological properties of strain YM-25 led to the conclusion that the microorganism was Streptomyces griseosporeus.

When the microorganism was aerobically cultured at 30°C in a jar fermentor containing the most suitable medium for growth which consisted of 5% glycerol, 0.5% polypepton, 0.2% meat extract, 0.1% yeast extract, 0.4% Na₂HPO₄ ? 12H₂O, 0.1% KH₂PO₄, and 0.05% MgSO₄ ? 7H₂O (pH 7.3), the highest activity of Haim was obtained on 23~26hr cultivation.

Haim had specific inhibitory activities against animal α-amylases but not against microbial and plant α-amylases.     

Desilicification from the High Silica Containing Kraft Black Liquor by the Improved Carbon Dioxide Method

Addition of NADH to crude but not to pure branched-chain α-keto acid decarboxylase decreased the CO₂ production from α-keto-β-methylvalerate (KMV) suggesting the presence of an NADH dependent inhibitor in the crude enzyme from Bacillus subtilis. This NADH-dependent decarboxylase inhibitor was purified to homogeneity by a fast protein liquid chromatography system.

The purified inhibitor was identical with leucine dehydrogenase as to N-terminal amino acid squence (35 residues) and molecular weight, and catalyzed the oxidative deamination of three branched chain amino acids (BCAAs), valine, leucine, and isoleucine. The decarboxylase inhibitor was therefore identified as leucine dehydrogenase. A decreased substrate availability caused by leucine dehydrogenase thus reasonably accounted for the NADH dependent inhibition of the decarboxylation. In turn, the observation that leucine dehydrogenase competes with the decarboxylase for branched-chain α-keto acid (BCKA) suggested an involvement of this enzyme in the branched chain fatty acid (BCFA) biosynthesis. This view was supported by the observation that addition of NAD to crude fatty acid synthetase increased the incorporation of isoleucine into BCFAs. Pyridoxal-5′-phosphate and α-ketoglutarate, cofactors for BCAA transaminase, modulated BCFA biosynthesis from isoleucine in vitro, suggesting also the involvement of transaminase reaction in BCFA biosynthesis.     

Inhibition of Trypanosoma cruzi α-Hydroxyacid Dehydrogenase-isozyme II by N-Isopropyl Oxamate and its Effect on Intact Epimastigotes

The effect of N-isopropyl oxamate on the activity of α-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.     

Characteristics of M-Gtfi,A New Inhibitor of Streptococcus Mutans Glucosyltransferase

Abstract

M-GTFI, originally screened as an inhibitor of Streptococcus mutans glucosyltransferase, strongly inhibited α-glucosidase, in a non-competitive manner especially when the synthetic substrate p-nitrophenyl-α-D-glucopyranoside was used. It also inhibited β-glucosidase, β-amylase and, to a lesser extent, β-glu-curonidase.

The inhibitor was stable in neutral and alkaline pH ranges and dependency of the inhibition on pH and temperature was not observed. Some proteinases and polysaccharides-hydrolyzing enzymes as well as human saliva did not inactivate the inhibitor.

There was a correlation between the release of sulfate anions from the inhibitor molecule on incubation with HCI (0.2 N) at 100°c and loss of inhibitory properties of the molecule. It is suggested that the presence of sulfate ester linkages in the inhibitor molecule play an important role in the inhibition process.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Isolation and Identification of Microorganism,Producing Microbial Alkaline Proteinase Inhibitor (MAPI)

In the screening of actinomycetes’ culture filtrate for inhibitor of subtilisin and various microbial alkaline proteinases, a novel inhibitor was found in a cultured broth of strain WT-27. This inhibitor was named as MAPI, abbreviation of microbial alkaline proteinase inhibitor.Judging from the morphological and physiological properties of the actinomycetes which produced MAPI, this strain was identified as Streptomyces nigrescens.

For the production of MAPI, this strain was aerobically cultured at 25 ~ 27%C in a jar fermentor which contained an optimum medium consisting of polypepton 3 %, meat extract 1%, glucose 1%, NaCl 0.1%, K₂HPO₄ 0.1% and MnSO₄·nH₂O 0.0001%, pH 7.0. The production of MAPI reached its maximum after 21 ~ 24 hr cultivation.

MAPI had an inhibitory activity against various microbial alkaline proteinases, α-chymotrypsin and papain but not against trypsin, kallikrein, thermolysin, or pepsin.     

The Biosynthesis and Metabolism of Acarbose in Actinoplanes sp. SE 50/110: A Progress Report

Abstract

The α-glucosidase inhibitor acarbose produced by Actinoplanes sp. SE50/110 is a pseudotetrasaccharide, which consists of an unsaturated cyclitol (carba-sugar), 4-amino-4,6-dideoxyglucose and maltose. The cyclitol (valienol) and the 4-amino-4,6-dideoxyglucose are linked via an N-glycosidic (imino) bond, forming the so-called acarviosyl moiety, which is primarily responsible for the inhibitory effect on α-glucosidases. The gene cluster encoding the biosynthetic genes for the synthesis of acarbose (acb-genes) was sequenced and 25 open reading frames belonging to the acb-gene cluster were identified. Based on the analysis of the enzymes encoded by the acb-cluster, the biosynthesis and ecological role of acarbose is described. The gene cluster includes genes which encode: proteins for the synthesis of the cyclitol; the enzymes for the synthesis of dTDP-4-amino-4,6-dideoxyglucose; glycosyltransferases for the condensation reactions; ATP-dependent exporters and importers; extracellular starch degrading enzymes; and intracellular acarbose modifying enzymes. Acarbose has a dual role for the producer: it inhibits α-glucosidic enzymes of competitors and functions as a carbophor for the uptake of glucose or starch molecules.     

Purification and Some Properties of Amylase Inhibitor (S-AI)

Amylase inhibitor (S-AI) was purified by about 25 times from culture filtrate of Streptomyces diastaticus subsp. amylostaticus No. 2476 through the methods of adsorption on active carbon, column chromatographies on Dowex 50 W × 2 (H-form) and Dowex 50 W × 2 (NH₄-form), gel filtration on Sephadex G-25, El-complex formation with BLA, isolation of complex by gel filtration on Sephadex G-75, dissociation from complex by the method of acid denaturation, rechromatographies on Dowex 50 W × 2 (NH₄-form) and Sephadex G-25. Homogeneity of this S-AI was examined by means of TLC, where S-AI gave a single spot in various solvent systems. S-AI specially inhibited α-amylases and glucoamylase, but not β-amylases and other glucoside hydrolases.

S-AI was a very stable substance, as it retained 100% of its original activity after being kept for 30 min at 100°C in a pH range between 3.0 and 10.0. The molecular weight of S-AI was estimated to be about 1500 by gel filtration on Sephadex G-15.

S-AI was regarded to be an oligosaccharide which was mainly composed of glucose in an amount of about 85 %. S-AI was hydrolyzed by β-amylase from non-reducing terminal and released two moles of maltose succesively.     

The Structure of a New Piperidine Derivative from Buckwheat Seeds (Fagopyrum esculentum Moench)

Streptomyces hygroscopicus No. B–5050-HA, which produces a mixture of six maridomycins, yielded a mutant which produced 75% of the mixture as maridomycin III (MDM III).

Growth of S. hygroscopicus No. B–5050-HA, an improved MDM producer, was almost completely inhibited by 20 µg/ml of valine. This inhibition was counteracted by the addition of isoleucine, threonine, homoserine, methionine, α-amino-n-butyrate and α-ketobutyrate.

A valine resistant mutant, strain AV was isolated and found to produce increased level of MDM III at the expense of other maridomycins. Production of MDM III by the parent strain depended on the addition of isoleucine to the medium, but that by this mutant did not.

The properties of strain AV were discussed.     

A Protease Inhibitor from Penicillium cyclopium

A protease inhibitor produced by Penicillium cyclopium on solid cultures of wheat bran was purified by means of column chromatography on Duolite A-2 and DEAE-cellulose, acetone precipitation and lyophilization. The purified inhibitor obtained as a white, floccose and hygroscopic substance was monodisperse by ultracentrifugal analysis. It was found to be an acidic macro-molecule of a molecular weight of about 5000. The chemical analyses rejected the possibility of the presence of amino acids, peptides, sugars, amino sugars, or uronic acids in the inhibitor molecule.

Properties of a protease inhibitor from Penicillium cyclopium were studied. The pH range of the inhibitor action is restricted to acid pH, optimally at pH 3. Increasing temperature accelerates its action upon enzyme. The inhibitor causes enzyme inactivation in proportion to its concentration. It is fairly stable in an acid solution but unstable in an alkaline solution. It undergoes destruction by heat, hydrogen peroxide and ascorbic acid. The inhibitor reversibly combines with Al³⁺, Fe³⁺, Ag⁺ and Cu²⁺ to produce a precipitate. Salts interfer with the inhibitor activity. Generally, acid proteases from various penicillia are susceptible to the inhibitor while those from other genera are resistant.     

Moranoline (1-Deoxynojirimycin) Fermentation and Its Improvement

Moranoline (1-deoxynojirimycin) is a strong α-glucosidase inhibitor. A rapid screening method for isolation of moranoline-producing Streptomyces (oblate agar plate method) was developed. A glucoamylase inhibitor was isolated from the culture filtrate of Streptomyces lavendulae GC-148, a mutant strain obtained from Streptomyces lavendulae MB-733, a soil isolate; its spectral data were identical with those of moranoline from Morus species.

Increased moranoline production was achieved through media improvement and mutagenic treatments (ultraviolet irradiation and N-methyl-N^′-nitro-N-nitrosoguanidine treatment): 27~33-fold augmentation (150 to 4000~5000 μg/ml) was obtained.     

Osservazioni prellminari sul meccanismo di azione della cocliobolina

Abstract

MECHANISM OF COCHLIOBOLIN ACTIVITY: A PREVENTIVE NOTE. — Coch-liobolin caused leakage of phosphate and organic compounds from corn rootlets and potato discs, at room temperature. The leakage does not occur at 1–5°C, but when potato discs are incubated at this temperature and then thoroughly washed and brought at 25°C, a sharp increase of phosphate may be noticed in the incubation solutions.

Cochliobolin inhibited partially the aerobic respiration of glucose and endogenous carbon in Micrococcus pyogenes var. aureus resting cells. Aerobic respiration of pyruvate, succinate, fumarate and malate was completely inhibited, while the inhibition of lactate and Lketoglutarate respiration required a short lag period. The hydrogen-ion concentration of the media seemed to be an important factor controlling the rapidity of action of the inhibitor, because at pH 4 and 5 at least 120 minutes were required prior any effect could be observed, while only 30 minutes were required at pH 6.

The effects of cochliobolin on Micrococcus resting cells were irreversible In contrast, respiratory activities of acetonic powders were refractory to the substance under aerobic conditions, and oxidation of pyruvate, malate, fumarate, succinate and α-ketoglutarate were not affected by saturated solutions of cochliobolin. It is suggested that the first site of attack is the cell wall-cell membrane unit, altering cell permeability, so that inorganic ions and other cofactors essential to respiration are lost in consequence of the leakage through the cell membrane.     

Studies on Biologically Active Substances Formed by Bacilli

The products of several Bacillus strains were investigated on rabbit serum calcium decreasing, oxytocic and toad heart function promoting activities. These products were obtained from the clear supernatant fluid of the culture medium after the cells were removed by centrifugation.

For the production of rabbit serum calcium decreasing substance, Bacillus subtilis K and Bacillus natto No. 8 were found to be usefull, Bacillus megaterium KM, Bacillus cereus var. mycoides and Bacillus subtilis K produced oxytocic principle. Bacillus subtilis K, Bacillus brevis and Bacillus megaterium KM also produced toad heart function promoting factor.

A procedure was developed to obtain the electrophoretically homogenous rabbit serum calcium decreasing substance from culture filtrate of Bacillus subtilis K. The crude substance was obtained as isoelectric precipitate by adjusting the culture filtrate to pH 3.0. The crude substance was purified by gel filtration on a Sephadex G-75 column, isoelectric fractionation and chromatography on DEAE-cellulose column. The purified preparation was shown to be homogenous by Tiselius electrophoresis but was separated into two bands by polyacrylamide electrophoresis. The chemical analysis of this biologically active substance indicated this substance to be a lipoprotein. The substance decreased rabbit serum calcium level about 12% at 6~8hr after intravenous injection (dose; 0.5 mg/kg body weight).     

A Convenient Method for Rapid Determination of Cereal Proteins: A Device to Eliminate the Effect of Color Substances on the Biuret Procedure

At least, four kinds of amylase inhibitors are found in culture of Streptomyces sp. No.280.¹⁾ A large amount of amylase inhibitors were produced by Streptomyces sp. No. 280 when cultivated on 3% oatmeal medium and it was found that the molecular weight of the inhibitors were transformed to smaller molecules during the cultivation time. The transformation of the amylase inhibitor was found to result from degradation of its carbohydrate moiety by α-amylase in the culture broth. The amylase inhibitor was hydrolyzed partially by the action of taka-amylase A or hog pancreatic α-amylase. With hydrolyzation of amylase inhibitor by α-amylase, neutral sugars (mainly maltose) were liberated from the amylase inhibitor and a modified inhibitor was newly formed, but amylase inhibitory activity against glucoamylase was not changed. The inhibitory activity against muscle Phosphorylase a, however, was almost completely lost.     

Cytinus hypocistis L. embryogenesis: Some biological and ultrastructural aspects of fertilization and embryo development

Abstract

The present paper examines some biological and ultrastructural aspects of fertilization and early development of the embryo in Cytinus hypocistis, a parasitic plant belonging to the Rafflesiaceae. The probable functions of a mucilaginous substance contained in the ovary and embedding the numerous pollen tubes coming from the style are discussed.

It was ascertained that pollen tubes pass through the micropyle and enter a synergid pushing, their way through the nucellar cells that show swollen walls owing to a probable enzymatic action whose function is to facilitate pollen tube penetration. It was hypothesized that the secretion of such enzymes is attributable to the numerous pollen present in the ovary or entering the mycropyle.

Since, in all the ovules observed, synergid degeneration was never found before the arrival of the pollen tube, this degeneration was interpreted as being caused by the material disharged by the pollen tube, rather than being an essential prerequisite for pollen tube penetration into the synergid.

Pollen tube content was observed to be made up of an intensely electron-dense substance surrounding many lipidic globules and numerous polysaccharide vesicles that fuse with the pollen tube wall, clearly contributing to its growth.

The sequence of the first divisions of the developing embryo was followed and the extreme reduction of the embryo is confirmed.

In Cytinus hypocistis starch is totally absent from all the sells belonging to the female gamethophyte as well as to those belonging to the embryo, but lipidic globules are very frequent; it is therefore supposed that these bodies constitute good material for the nutrition of the zygote and early embryo.     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of investigation of alicyclic hydrocarbon-utilizing microorganisms, five strains of ethylcyclohexane-utilizing bacteria were isolated from soil samples.

Among those bacteria, the strain S6B1 that was identified as Alcaligenes faecalis, showed the best growth in shaking culture.

The strain S6B1 was found to produce 4-ethylcyclohexanol from ethylcyclohexane.

This substance separated from culture broth was purified and identified to be trans-4-ethylcyclohexanol by the use of NMR.     

Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics

Abstract

The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein–ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC₅₀ was measured (IC₅₀ = 1.738?±?0.041?mM), and the dissociation constant was determined (K_{i Slope} = 0.46?±?0.04?mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. Abbreviations AKG alpha-ketoglutaric acid

pNPG 4-nitrophenyl-α-d-glucopyranoside

ANS 1-anilinonaphthalene-8-sulfonate

MD molecular dynamics.

Communicated by Ramaswamy H. Sarma     

An α-1, 3-Glucanase from Streptomyces sp. KI-8 Production and Purification

An α-l,3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-l,3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-l,3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.

The enzyme was purified by (NH₄)₂SO₄ precipitation, column chromatography on DEAE-cellulose and P(phospho)-cellulose. To eliminate the concomitant β-l,3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-l,3-glucanase was eluted with a solution of α-1,3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78,000 by SDS-polyacrylamide gel electrophoresis.     

Effects of aldehydes on CD36 expression

Introduction: During the oil frying process lipid peroxidation compounds are formed. These products can modulate gene expression and alter cellular behaviour. The cellular uptake of oxidized LDL, a key step in the development of atherosclerosis, is mediated by the CD36 scavenger receptor, whose expression is down-regulated by α-tocopherol.

Objective: To determine the effects of water-soluble aldehydes, obtained from thermally oxidized sunflower oil on the expression of CD36 scavenger receptor in human monocytes (THP-1 cells). We also wanted to study the effects of α-tocopherol on CD36 expression in the presence of water-soluble aldehydes.

Materials and Methods: Sunflower oil was heated in a frying pan, at 180–200°C for 40?min, water-soluble aldehydes were isolated, and the content of thiobarbituric acid reacting substances (TBARS) was determined. THP-1 monocytes were cultured in RPMI medium during 24?h and incubated with increasing concentrations of the water-soluble aldehydes (ranging from 0.05 to 1?μM) and with or without 50?μM of α-tocopherol. In parallel, THP-1 cells were cultured with the same volume of an extract obtained from non-oxidized oil or distilled water. The CD36 expression at the cell surface was studied with fluorescence-activated cell sorting (FACS).

Results: Monocytes incubated in a medium containing water-soluble aldehydes, showed a dose dependent increase in the expression of the CD36 protein on the cell surface, compared to with the control groups. When the cells were treated simultaneously with 50?μM of α-tocopherol a significant reduction in the expression of the CD36 protein was observed.

Conclusion: Water-soluble aldehydes, extracted from thermally oxidized culinary oil, increase the expression of CD36. This effect is partially decreased by the presence of α-tocopherol.