Studies on Barley and Malt Amylases

From the 2 m urea extract of ground barley two zymogen β-amylase (Z-β-A) fractions (Fractions A and B) and one active β-amylase (A-β-A) fraction (Fraction C) were isolated by Sephadex G-75 gel filtration and purified by DEAE-Sephadex A-50 column chromatography. The molecular weights of Fractions A, B and C were estimated to be approximately 280,000, 160,000 and 56,000, respectively.

Both the Z-β-A fractions which were ultracentrifugally and electrophoretically homogeneous were found to be accompanied by a small amount of saccharogenic activities. From the estimation of Km values for these saccharogenic activities and the behavior in their activation with 2-mercaptoethanol and papain, it seems reasonable to conclude that Fraction A is a heteropolymer type of Z-β-A composed of both A-β-A and barley reserve proteins; that Fraction B is a homopolymer type of Z-β-A composed of A-β-A alone; and that two different activation mechanisms, proteolytic activation and disulfide bond cleaving activation, are necessary for full activation of Z-β-A in barley.     

Studies on Pectic Enzymes of Microorganisms

Endo-polygalacturonase (endo-PG) of Aspergillus saitoi was purified through ammonium sulfate fractionation, Amberlite IRC-50 column chromatography, and several combinations of Sephadex column chromatography.

The purified endo-PG, which was almost homogeneous ultracentrifugally and electrophoretically, had the sedimentation constant of 2.2 S and the absorption maximum at 277 mμ. Its optimum pH and temperature were 4.8~5.0 and 45°C, respectively, and it was most stable between pH 4.0 and 6.0, but over 90% of the activity was lost at 50°G for 10 min.

The purified enzyme was a typical endo-PG, and hydrolyzed about 60% and 17% of glycosidic linkage of polygalacturonic acid and pectin, respectively. This enzyme preparation had no pectinesterase, trans-eliminase, and apple juice-clarifying activities, but macerated potato tuber slices singly.     

Purification,Crystallization and Some Properties of Lipase from Geotrichum candidum Link

Lipase (EC 3.1.1.3) of Geotrichum candidum Link was purified by means of ammonium sulfate fractionation, DEAE-Sephadex column chromatography, gel-filtration on Sephadex G–100 and Sephadex G–200, and was finally crystallized in concentrated aqueous solution. It was confirmed that the crystallized preparation was homogeneous electrophoretically and ultracentrifugally.

It was estimated with the crystalline enzyme that the sedimentation constant (s₂₀, w) was 4.0, the isoelectric point was pH 4.33, and the molecular weight was 53,000~55,000. From the result of amino acid analysis, none of sulfur containing amino acid was detected in the enzyme. It was also recognized that the crystalline preparation contained about 7% of the carbohydrate and very small amount of lipid. It was characterized that the lipase was the most active at pH 5.6~7.0 on olive oil, at 40°C and was stable in the range of pH 4.2 to 9.8 at 30°C for 24 hr, and was stable below 55°C for 15 min.     

Preparative-scale isolation and purification of procaryotic and eucaryotic ribosomal 5 S RNA: Bacillus subtilis, Neurospora crassa, and wheat germ

Ribosomal 5 S RNA from three different organisms has been isolated in high yield and purity. Without prior isolation of ribosomes, a presoak in buffer followed by phenol extraction, DE-32 ion-exchange chromatography, and Sephadex G-75 gel-permeation chromatography yields at least 5-10 mg of electrophoretically homogeneous 5 S RNA from 100 g of cells. Ribonuclease activity is eliminated by various combinations of low temperature, sodium dodecyl sulfate, phenol, and bentonite. High-molecular-weight contaminants are suppressed by either 65 degrees C heat treatment or lowered sodium dodecyl sulfate concentration. For the eucaryotes, 5.8 S RNA contamination is reduced either by low temperature in the initial solubilization or by postponing 65 degrees C heat treatment until after the phenol extraction step.     

Studies on the Non-starchy Polysaccharides of the Endosperm of Buckwheat

Water soluble polysaccharides from the buckwheat endosperm was fractionated by salting out and a DEAE-cellulose column (phosphate form) chromatography and the main component (polysaccharide A₁) was isolated as an ultracentrifugally and electrophoretically pure preparation.

The content of polysaccharide A₁ in the buckwheat endosperm was 0.1~0.2%.

Its water solution showed high viscosity and [α]_d was +39.4°. The molecular weight was 240,000~260,000.

Polysaccharide A₁ consisted of xylose, mannose, galactose and glucuronic acid. The hydrolysis of methylated polysaccharide A₁ gave 2,3,4-tri-O-methyl-xylose, 2,3,4,6-tetra-O-methyl-galactose, 2,4,6-tri-O-methyl-galactose, di-O-methyl-mannose and 4-O-methyl- and 5-O-methyl-glucuronic acid. These results suggested that the main chain of this polysaccharide consisted of glucuronic acid, mannose and galactose and the former two occupied branching position with xylose and galactose residues as nonreducing end.     

Purification and Characterization of Aminopeptidase from Euphausia superba

Krill aminopeptidase was purified about, 1,100-fold from an extract of Euphausia superba with DEAE-cellulose column chromatography, Toyopearl HW55, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was determined to be 140,000 by gel filtration and SDS-polyacrylamide disc gel electrophoresis. The optimum pH and optimum temperature were 8.4 and 45°C respectively. Krill aminopeptidase was inhibited by EDTA, Hg^{+ +} and amastatin, and partially by bestatin, and was activated by Co ^{+ +}. Alanyl-p-nitroanilide was hydrolyzed faster than leucyl-p-nitroanilide. Alanyl peptides (di-, tri-, tetra- and hexa-alanyl peptide) were hydrolyzed very fast.

These results suggest that krill aminopeptidase is an alanine aminopeptidase which is activated by cobaltous ion.     

Diagnosis of <Emphasis Type="Italic">Chenopodium album</Emphasis> allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.     

Morphogenesis of the tail fiber of bacteriophage T4

Summary The tail fiber component ofcoli phage T4 was purified and partially characterized. The material was purified approximately 1 200 fold over the original lysate obtained fromE. coli B/1 cells infected with a mutant in gene 34 (am A455). The purified material was ultracentrifugally, electrophoretically and electron microscopically homogeneous. Its chemical composition were also analyzed.The purified component was characterized to be a half fiber controlled by at least four genes, 35, 36, 37, and 38.This work was supported by a grant from the Ministry of Education, Japan, and a grant (No. 5 ROI GM-10982) from the National Institute of Health, U.S.A.     

3种不同油桐种仁蛋白质提取方法比较研究

一种有效的蛋白质提取方法是蛋白质双向电泳成功分离的关键。油桐种仁可以用来制取工业用桐油,但其中富含大量的能干扰蛋白质提取的物质,并能影响双向电泳图谱的分辨率。本文中对油桐种仁蛋白质采用丙酮提取（方法A）、苯酚抽提（方法B）以及丙酮—苯酚结合提取（方法C）,并通过蛋白质双向电泳技术对这3种方法的提取效果进行比较研究。结果显示：采用方法C所提取的蛋白质样品,其蛋白浓度高达到8.1 μg·μL^-1;并且该方法对油桐种仁中的高分子量、低分子量蛋白均有较强的提取能力;此外,其蛋白样品经过双向电泳所得到的蛋白质点和图谱分辨率也较其余两种方法好。     

Purification and some enzymatic properties of the chitosanase from Bacillus R-4 which lyses Rhizopus cell walls.

A strain of Bacillus sp (Bacillus R-4) produces a protease and a carbohydrolase both of which have the ability to lyse Rhizopus cell walls. Of the enzymes, the carbohydrolase has been purified to an ultracentrifugally and electrophoretically homogeneous state, and identified as a chitosanase. The enzyme was active on glycol chitosan as well as chitosan. Molecular weight of the purified enzyme was estimated as 31 000 and isoelectric point as pH 8.30. The enzyme was most active at pH 5.6 and at 40 degrees C with either Rhizopus cell wall or glycol chitosan as substrate, and was stable over a range of pH 4.5 to 7.5 at 40 degrees C for 3 h. The activity was lost by sulfhydryl reagents and restored by either reduced glutathione of L-cysteine. An abrupt decrease in viscosity of the reaction mixture suggested an endowise cleavage of chitosan by this enzyme.     

Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5-HT3 Receptor-Mediated Cation Permeability in NG 108-15 Cells

Abstract: In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5-hydroxy-tryptamine (5-HT)] and substance P induces the opening of a cation permeability monitored by the influx of [¹⁴C]-guanidinium. The serotoninergic component of this cation permeability Is linked to 5-HT₃ receptor activation, whereas the substance P component probably involves an “N-terminal-dependent substance P receptor.” In this study, [¹⁴C]guanidinium influx triggered by 1 μM 5-HT plus 10 μM substance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω-conotoxin, as expected from a process independent of voltage-sensitive sodium and calcium channels. In contrast, [¹⁴C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis-O-aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [¹⁴C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [¹⁴C]guanidinium uptake was markedly increased even in the absence of 5-HT and/or substance P. Conversely, [¹⁴C]guanidinium influx due to the latter substances could be reversibly and dose-dependently blocked by various drugs that possess calmodulin-antagonizing properties. These results strongly suggest that the cation permeability opened by 5-HT and substance P in NG 108–15 cells involves a calcium/calmodulin-dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12-O-tetradeca-noylphorbol 13-acetate did not alter [¹⁴C]guanidinium influx in NG 108–15 cells exposed to 5-HT and substance P., this process probably does not involve the calcium-dependent nitric oxide pathway and protein kinase C activation.     

Phenolic removal of olive-mill dry residues by laccase activity of white-rot fungi and its impact on tomato plant growth

We studied the influence of the laccase activity of two white-rot fungi on the toxic effect of water-soluble substances from dry residues of olives (ADOR) on tomato plants. Pycnoporus cinnabarinus and Coriolopsis rigida decreased the phenol content of ADOR to 73% after 15 days. P. cinnabarinus and C. rigida produced laccase activity after 5 and 15 days, respectively, and the highest activity in both fungi was detected at 20 days. The treatment of ADOR with these white-rot fungi decreased the phytotoxicity of this residue on tomato plants. A close relationship was found between the amount of laccase produced, the decrease in phenol content of ADOR by the saprobic fungi, decrease of phytotoxicity of ADOR, and the increase in dry weight of tomato plants. These results show that phenol removal by the laccase activity of white-rot fungi can be important in the elimination of phytotoxic substances present in olive-mill dry residues.     

Acyclonucleoside Inhibitors of Uridine Phosphorylase

Abstract

Various acyclonucleoside analogues have been examined as inhibitors of highly purified, and electrophoretically homogeneous, bacterial uridine phosphorylase.     

A Red,Fluorescent Protein from Silkworm

Nondiffusable melanoidin obtained from a glycine-xylose system was heated in aqueous media, and the resulting chemical changes, as affected by the presence of oxygen, pH value, temperature and the addition of transitional metals, were investigated.

Melanoidin, when heated at 90°C in an aqueous solution, caused remarkable discolorization accompanied by the development of fluorescence, oxygen consumption and a noticeable variation of reductone content. Heated melanoidin became polydispersive in molecular weight on gel filtration chromatograms. There appeared reductones, ninhydrin-positive substances, fluorescent substances, aromatic amines, aliphatic carbonyls, and aliphatic primary amines and/or methylene groups in diffusâtes of melanoidin heated in various media.

An increase in pH value favored oxidative discolorization, while an increase in the concentration of transitional metals except Mn²⁺ restrained the discolorization. In the absence of oxygen, heated melanoidin brought about a slight strengthening of color and the accumulation of reductones ca. fifteen times more than the initial level, while in the presence of oxygen the increase of reductone content at the early period was followed by a rapid decrease.

According to the results obtained, the ambivalent reactivity of melanoidin, i.e. polymerization (colorization) and depolymerization (discolorization), was discussed in relation to influencing factors. A mechanism for the production of reductones in heated melanoidin was also proposed.     

Preparation and immunochemical investigation ofShigella polysaccharide labelled with32P

Preparation of theShigella flexneri 2a lipopolysaccharide labelled with³²P is described. For the extraction we used a mixture of phenol and water (1∶1) at 65–69°C. Ballast substances were removed by precipitation with trichloracetic acid and by ethanol. The preparation was checked by agar electrophoresis. Ouchterlony's diffusion and immunoelectrophoresis. Its behaviour during gel filtration on a column of immunosorbent prepared by binding immune gamma globulin to Sephadex G-25 modified chemically by introducing a CN(-) group through the action of cyanogen bromide, was studied.     

Purification and Properties of α-Glucosidase from Bacillus cereus

α-Glucosidase has been isolated from Bacillus cereus in ultracentrifugally and electrophoretically homogeneous form, and its properties have been investigated. The enzyme has a sedimentation constant of 1.4 S and a molecular weight of 12,000. The highly purified enzyme splits α-d-(1→4)-glucosidic linkages in maltose, maltotriose, and phenyl α-maltoside, but shows little or no activity toward polysaccharides, such as amylose, amylopectin, glycogen and soluble starch. The enzyme has α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme can also catalyze the transfer of α-glucosyl residue from maltose to riboflavin. On the basis of inhibition studies with diazonium-1-H-tetrazole, rose bengal and p-chloromercuribenzoate, it is assumed that the enzyme contains both histidine and cysteine residues in the active center.     

Purification of glucose isomerase from Streptomyces nigrificans

Summary A relatively simple method for obtaining an electrophoretically homogeneous preparation of glucose isomerase from Streptomyces nigrificans is described. Extract of disintegrated microbial cells was first heated at 60°C in the presence of Co and Mg ions. Centrifugation and ultrafiltration were followed by ion exchange chromatography on DEAE-cellulose. The fraction with glucose isomerase activity proved to contain no proteins other than the isolated enzyme.     

Constituents of Japanese Peppermint Oil

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121 °C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20 d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.     

Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

Pollen Tube Growth Inhibitors Involved in the Ovary of Self-Incompatible Japanese Pear

The growth inhibitors of pollen tubes in the pistils of Japanesepear ‘Chojuro’ were studied in vitro to elucidatethe physiological mechanism of self-incompatibility. Addition of water extracts from ovaries into a well dug in anagar medium containing sucrose and boric acid inhibited thegrowth of incompatible pollen tubes more strongly than thatof compatible ones. The substance, tentatively called Sinhibitor(self-inhibitor), was detectable in a relatively wide rangeof concentrations of the extract, from 10- to 60-fold dilution.However, it was absent in the stylar extract. S-inhibitor was stable, even though the extract was heated to100?C for 10 min. The chromatogram of the S-inhibitor followinga Sephadex G-10 gel filtration showed 2 peaks of inhibition:one peak corresponded to the peaks of protein and phenol (phenolsmay be conjugated with proteins), and the other to that of reducingsugar. The water extract from mature ovaries when diluted 10-fold wasa stronger growth inhibitor of incompatible pollen tubes comparedwith that from immature ones. This substance, tentatively calledA-inhibitor (adult-inhibitor), appeared to be different fromS-inhibitor. (Received May 20, 1986; Accepted December 22, 1986)