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.     

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.     

Modification of an arginine residue of a base-nonspecific ribonuclease from Aspergillus saitoi.

1. A base-nonspecific ribonuclease from Aspergillus saitoi [RNase Ms, EC 3.1.4.23; molecular weight, 12,500] was modified with phenylglyoxal (PG) and 1,2-cyclohexanedione (CHD) in order to determine whether a single arginine residue was involved in the active site of the enzyme. 2. RNase Ms was inactivated by both PG and CHD with concomitant loss of one arginine residue. A competitive inhibitor of RNase Ms, 2',(3')-AMP, protected the enzyme from inactivation by PG. These findings strongly suggest that one arginine residue is involved in the active site of RNase Ms. 3. Difference CD spectra were measured at pH 5.5 for the binding of 2'-AMP and adenosine to native RNase Ms and the CHD- and PG-modified enzyme derivatives to determine the association constants. The arginine modification brought about a marked decrease in the binding affinity of 2'-AMP for the enzyme, but only a slight decrease for adenosine, suggesting that the arginine residue had interacted with the phosphate groups of the substrate.     

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.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylendopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococcal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26,596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the active sites of these RNases.     

Purification and properties of a phospholipase C that has high activity toward sphingomyelin from Aspergillus saitoi

An enzyme hydrolyzing sphingomyelin was purified from extracts of solid cultures of Aspergillus saitoi 7041 by fractionation with isopropanol followed by successive column chromatographies on DEAE-Sepharose CL-6B, butyl-Toyopearl 650 M, and phenyl-Sepharose CL-4B. The preparation of purified enzyme was homogeneous and had an activity increased 81-fold over that of the isopropanol fraction. The yield was about 65%. The molecular weight was estimated to be 54,000 by sodium dodecyl sulfate-gel electrophoresis. The enzyme solution had a violet color and contained iron atoms. The enzyme catalyzed the hydrolysis of sphingomyelin to N-acylsphingosine and phosphorylcholine. The optimum pH for hydrolytic activity was around 3.5. The Km values for sphingomyelin and 2-hexadecanoylamino-4-nitrophenylphosphorylcholine were 0.11 and 0.33 mM, respectively. The enzyme also catalyzed the hydrolysis of other phospholipids; the order of its hydrolytic activity at a substrate concentration of 2.5 mM was phosphatidylcholine greater than or equal to sphingomyelin = phosphatidylethanolamine = lysophosphatidylethanolamine greater than phosphatidyl DL-glycerol = phosphatidyl L-serine greater than phosphatidylinositol. From these results, this enzyme appears to be a new type of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3).     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

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.     

Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.     

Characterization of two forms of base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

Two forms of RNases (RNase ML and RNase MM) from Aspergillus saitoi which are base non-specific and adenylic acid preferential were separated from each other by DEAE-cellulose column chromatography. They are indistinguishable with respect to enzymatic properties such as base preferability, pH optimum, kinetic constants measured with 2',3'-cUMP and 2',3'-cCMP as substrates, and effects of ionic strength, physical properties such as heat stability, isoelectric point and circular dichroism spectra, amino acid composition and immunological property. They only differ in carbohydrate content. The apparent molecular weight determined by SDS-disc electrophoresis was 36,000 for RNase ML and 32,000 for RNase MM. Both RNases were reduced and carboxymethylated, and then digested with trypsin, separately. Glycopeptides were isolated from the both digests by gelfiltration and paper chromatography. The amino acid compositions of glycopeptides obtained from RNase ML (ML TS-IIC) and that obtained from RNase MM (MM TS-IIIC) were the same. The amino acid sequences of both glycopeptides determined by Edman degradation and carboxypeptidase digestion were also the same. The results indicated that RNase ML and RNase MM were the same protein having different sizes of carbohydrate chains at one site on the molecule.