Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.     

Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi

An alpha-mannosidase differing from 1,2-alpha-mannosidase was found to occur in Aspergillus saitoi. By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.     

黑曲霉纤维素酶的纯化及酶学性质研究

黑曲霉(Aspergillusniger)固态发酵后粗酶液经硫酸铵盐析,2次SephadexG-200柱层析后可提纯8倍左右.CMC酶最适作用温度为60℃,最适作用pH为3.5,30℃～70℃区间酶活力较稳定,在pH3.0～5.0范围内,50℃保温30min能保持80%的酶活力.CMC酶的Km、Vmax值分别为7.69%CMCg/ml、0.33mg/ml·     

N-bromosuccinimide oxidation of a glucoamylase from Aspergillus saitoi

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with NBS was studied. 2. The tryptophan residues in Glu M1 were oxidized at various NBS/Gluc M1 ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M1 with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Glu M1, the enzymatic activity of Gluc M1 decreased to almost zero (NBS/Gluc M1 = 20). Thus, the most essential tryptophan residue(s) is amongst these 4-5 tryptophan residues. 3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M1 = 50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant. 4. In the presence of 10% soluble starch, about 5 tryptophan residues in Gluc M1 were oxidized at an NBS/Gluc M1 ratio of 20. The remaining activity of Glu M1 at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue(s) is amongst these 2 tryptophans. 5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M1 showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M1 and decreased with NBS oxidation.     

Regulation of phospholipase C delta activity by sphingomyelin and sphingosine.

Phospholipase C delta (PLC delta) is strongly inhibited by sphingomyelin (SM). The inhibition occurs in both the presence and the absence of spermine, an activator of PLC delta. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) also inhibit PLC delta in the presence of spermine but are much less effective than SM. PE and PC activate and PS and PI inhibit PLC delta in the absence of spermine. Again, the inhibition by PS and PI is much weaker than the inhibition observed with SM. Similar or identical effects are observed in detergent micelle and liposome assays. Comparisons of physiological concentrations of SM with concentrations yielding 50% inhibition of PLC delta in vitro indicate that SM is likely to be a major factor in regulating the activity of PLC delta by inhibition. It is proposed that, in vivo, sphingomyelin acts as an inhibitor of PLC delta, which enables the enzyme to be regulated by activation. In certain circumstances, there is a substantial decline in SM and this may lead to a partial relief of the inhibition. PLC delta is activated by sphingosine in the absence of spermine. However, this activation occurs at unphysiologically high concentrations of sphingosine. The effects of SM and sphingosine on PLC delta in marked contrast to those observed with protein kinase C, which is unaffected by sphingomyelin and inhibited by sphingosine.     

Carboxymethylation of a minor ribonuclease from Aspergillus saitoi.

(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.     

Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity.

The specificity and mode of action of an acid proteinase (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N,O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24-)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, leucine and isoleucine in the P'1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogenactivating acid proteinase from Asp. saitoi.     

Phytophthora infestans has a plethora of phospholipase D enzymes including a subclass that has extracellular activity

In eukaryotes phospholipase D (PLD) is involved in many cellular processes. Currently little is known about PLDs in oomycetes. Here we report that the oomycete plant pathogen Phytophthora infestans has a large repertoire of PLDs divided over six subfamilies: PXPH-PLD, PXTM-PLD, TM-PLD, PLD-likes, and type A and B sPLD-likes. Since the latter have signal peptides we developed a method using metabolically labelled phospholipids to monitor if P. infestans secretes PLD. In extracellular medium of ten P. infestans strains PLD activity was detected as demonstrated by the production of phosphatidic acid and the PLD specific marker phosphatidylalcohol.     

Esterolytic Activity of Acid Carboxypeptidase from Aspergillus saitoi

Asymmetric reduction of aromatic ketones using chirally modified reagents prepared from sodium borohydride and optically active acids in the presence or absence of 1,2: 5,6-di-O-isopropylidene-α-d-glucofuranose produced the corresponding optically active alcohols with optical yields of 4 ~ 47%. The reagent prepared from sodium borohydride and 1 equivalent of l-malic acid in the presence of 2 equivalents of 1,2: 5,6-di-O-isopropylidene-α-d-gluco-furanose gave the highest yields.     

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.     

Purification of phospholipase C isoenzyme 1 from Clostridium perfringens and its properties

A procedure for the purification of isoenzyme I of phospholipase C from Cl. perfringens was developed. The isoenzyme was purified to homogeneity (data from disc electrophoresis) using affinity chromatography on polycephamide and gel filtration through Ultrogel AcA-54, the enzyme yield being 41%. Some properties of the purified isoenzyme I (pH and temperature optima, stability, effect of metal ions and detergents, substrate dependence) were investigated. No significant differences between the properties of the unfractionated enzyme and isoenzyme I were established.     

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.     

Purification of a phosphoinositide-specific phospholipase C from bovine brain

A soluble phosphoinositide-specific phospholipase C (PLC) was purified 58,000-fold from bovine brain. The enzyme, one of six distinct PLC activities detected in brain, accounted for approximately 15% of the soluble phosphatidylinositol-4,5-bisphosphate-phospholipase C (PIP2-PLC) activity in this tissue. The purification scheme included hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on phosphatidylinositol-Sepharose (PI-Sepharose). The enzyme was specifically eluted from the PI-Sepharose with PI, calcium, and detergent. The purified PLC had an estimated molecular weight of 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaved as a monomeric protein during sedimentation on glycerol gradients. The enzyme required calcium for activity, exhibited a pH optimum of 6.5, and cleaved only phosphoinositides. The rates of PIP2 and phosphatidyl-4-monophosphate hydrolysis exceeded the rate of PI hydrolysis under all conditions tested. These properties are consistent with a potential role for this PLC in the early events involved in cellular calcium mobilization.