Purification and Characterization of Xylanases from Alkalophilic Thermophilic Bacillus spp.

Xylanases from alkalophilic thermophilic Bacillus spp. Wl and W2 were purified and characterized. The xylanases from the two strains were fractionated into two active components (I and II) by DEAE-Toyopearl 650M chromatography. Components I from the two strains had similar properties: optimum pH, 6.0; optimum temperature, 65°C; isoelectric point, pH 8.5 and 8.3; molecular weight, 21,500 and 22,500; and Michaelis constant, 4.5 and 4.0mg-xylan/ml. Components II from the two strains also had similar properties: optimum pH, 7.0~9.0 and 7.0~9.5; optimum temperature, 70°C; isoelectric point, pH 3.6 and 3.7; molecular weight, 49,500 and 50,000; and Michaelis constant, 0.95 and 0.57mg-xylan/ml. The activities of components I and II were inhibited by Hg⁺⁺ and Cu⁺⁺. Components I hydrolyzed xylan to yield xylobiose and higher oligomers, but components II produced xylose other than xylobiose and xylooligomers.     

Characterization of an Aspartic Proteinase of Mucor pusillus expressed in Aspergillus oryzae

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1–2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo-β-N-acetyl-glucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.     

Characterization of an Aspartic Proteinase of Mucor pusillus expressed in Aspergillus oryzae

The aspartic proteinase (MPP) gene from the zygomycete fungus Mucor pusillus was introduced into an ascomycete fungus, Aspergillus oryzae, by protoplast transformation using the nitrate reductase (niaD) gene as the selective marker. Southern blot analysis indicated that the MPP gene was integrated into the resident niaD locus at a copy number of 1–2. MPP secreted by the recombinant A. oryzae was correctly processed but was more highly glycosylated than that produced in the original M. pusillus strain. Treatment with endo--N-acetyl-glucosaminidase H and analysis of the carbohydrate composition of the secreted MPP revealed that the extra glycosylation of the MPP secreted by the recombinant A. oryzae was due to altered processing of mannose residues. The extra glycosylation of MPP affected its enzyme properties including its milk-clotting and proteolytic activities.     

Metabolism and Transport of [2-14C]({+/-}) Jasmonic Acid in the Potato Plant

Metabolism and transport of [2-¹⁴C](±) jasmonic acid(JA) were studied using 2-week-old potato plants. Plants grownunder tuber inducing conditions (short days) and non-inducingconditions (long days) were assayed 1, 5, 10, 15, 20, and 25days after substance application. The incorporated [2-¹⁴C](±)JAwas metabolized to tuberonic acid glucoside (TAG) within 2 weeks.The radioactivity of the ethanol extract from each plant partwas measured. More than half of the total radioactivity wasstill associated with the treated leaves up to 25 days afterthe application of [2-¹⁴C](±)JA. In the other parts ofthe plants, the upper leaves contained the most total radioactivity,followed by the stems and lower leaves. Under long days no tuberswere produced, and the radioactivity of the stolons was observed.In contrast, under short days tuber were produced, and the radioactivitieswere present in stolons and tubers. Flower buds and flowerswere produced more under long days and accumulated high radioactivity.It is concluded from the combined data that JA is metabolizedto TAG and transported to all parts of the plant. A high accumulationof TAG in tubers and flower buds may induce the formation oftubers and flower buds. (Received January 22, 1996; Accepted April 15, 1996)     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

The confronting cisternae in neurons in the substantia gelatinosa of the spinal cord of rats and Japanese monkeys

The neurons in the substantia gelatinosa of the spinal cord of the rat and Japanese monkey were investigated electron microscopically and ultracytochemically. The confronting cisternae observed in the cytoplasm of the majority of gelatinosal neurons in both species were composed of closely apposed parallel cisternae with electron-dense flocculent material, and a continuity with the cisternae of the rough endoplasmic reticulum was often observed. Ultracytochemically, the p-nitrophenylphosphatase (p-NPPase) activity was present in the confronting cisternae, but thiamine monophosphatase (TMPase) and thiamine pyrophosphatase (TPPase) activities were absent. These results indicate that the confronting cisternae are a variant of the rough endoplasmic reticulum.     

Ultraviolet resonance Raman and absorption difference spectroscopy of myoglobins: titration behavior of individual tyrosine residues

The UV resonance Raman spectra of horse and sperm whale myoglobin excited at 240 nm show bands between 600 and 1700 cm-1 which derive from tyrosyl and tryptophyl residues. No significant contribution from phenylalanine and peptide backbone vibrations occurs at this excitation wavelength. We examine the pH dependence of the UV resonance Raman and UV absorption difference spectra of these myoglobins to correlate the local protein environment of the tyrosyl residues as given by the protein crystal structure to their pKa values, molar absorptivities, and Raman cross sections. Some of our pKa values for the tyrosinate residues of horse Mb differ from those of previous studies. We show that the lambda max values, the molar absorptivities, and the Raman cross sections are sensitive to the local environment of the tyrosinate residues in the protein. We relate differences in the tyrosyl absorption spectra to differences in Raman cross sections. In addition, we discuss the importance to the Raman cross sections of the local electromagnetic field enhancement due to the dielectric environment of the tyrosinate residues in the protein. This local field should scale the Raman cross sections in a way useful as a probe of the average aromatic amino acid residue environment.     

rig encodes ribosomal protein S15. The primary structure of mammalian ribosomal protein S15

rig, a gene originally isolated from a rat insulinoma cDNA library, codes for a basic 145 amino acid protein [( 1986) Diabetes 35, 1178-1180]. Here we show that the immunoreactivity to a monoclonal antibody against the deduced rig protein and the translation product of rig mRNA comigrated with ribosomal protein S15. The amino acid sequence of ribosomal protein S15 purified from rat liver coincided with that deduced from the nucleotide sequence of rig mRNA, but there were indications that the initiator methionine was removed and the succeeding alanyl residue was monoacetylated. From these results, we conclude that the product of rig is ribosomal protein S15.     

Repression of nitrate reductase in cucumber leaves caused by calcium deficiency

Growth of young cucumber plants was strongly inhibited, whencalcium was removed from the culture solution. The activitiesof nitrate reductase, glutamate dehydrogenase and glutaminesynthetase were investigated after the removal of calcium. Thoughthe activities of glutamine synthetase and glutamate dehydrogenasewere not altered much, nitrate reductase activity, measuredby in vitro and in vivo assays, decreased dramatically. Theloss of nitrate reductase activity coincided with the levelof nitrate in the leaves. When nitrate was supplied to the cucumberswith a nitrate deficiency, the plants induced nitrate reductasetogether with a distinct accumulation of nitrate. However, cucumberstreated for both calcium and nitrate deficiency failed to inducenitrate reductase and to accumulate nitrate on the additionof large amounts of nitrate. Leaf sections that had been treatedfor both calcium and nitrate deficiency could induce nitratereductase when floated on nitrate solution under the light.This indicates that the drastic loss of nitrate reductase causedby the removal of calcium was due mainly to the deficiency ofnitrate as the inducer in leaves. (Received December 19, 1979; )