Detection and serological relationships of cymbidium mosaic potexvirus isolates.

Twenty-two isolates of Cymbidium mosaic virus (CyMV) were isolated from 35 orchid plants suspected of being infected with CyMV. Among the three methods used for detecting CyMV, immunoelectron microscopy (IEM-1) was shown to be the most sensitive method, being able to detect the virus in 71.43% of suspected CyMV-infected plants while the electron microscopic method and the indexing plant method could detect 51.43 and 42.86%, respectively. Out of 12 symptomless plants investigated, 25% were found by IEM-1 method to be infected with the virus. Purified CyMV were flexuous rods having lengths between 470-490 nm. A few end-to-end aggregates were also observed and the 280 260 absorbance ratios were from 0.884 to 0.929. The yield of CyMV was 31.07 to 44.09 mg per kg of Datura leaves. Antibodies against purified CyMV D2 were produced in rabbits and hens. The antibody titers in the yolk and sera of hens indicated that 0.5 mg of virus per immunization efficiently generated an abundant supply of IgY in the yolk, however 1 mg of virus per immunization gave a stronger immune response in both sera and yolk. The average yields of IgY were 6.5 +/- 0.6 and 9.4 +/- 0.9 mg/ml of yolk in the group that received 0.5 mg and the group that received 1.0 mg of the virus, respectively. Positive ELISA reactions were observed in 18 and 20 of 22 CyMV isolates when detected with rabbit IgG and IgY, respectively, demonstrating that those isolates were serologically related and the ELISA reactions were shown to be stronger with IgY than those with rabbit IgG in most isolates. The degree of reaction between the CyMV isolates, O(2) and O(4), and IgY was less than that of the other isolates. The two isolates, D(6) and Cat(6), gave negative reactions to rabbit IgG. The results of ELISA assays showed that the homologous serological reaction was not consistently stronger than the heterologous one. Twelve isolates out of twenty-two gave stronger reactions than the homologous antigen (CyMV D(2)) when IgY was used as the detecting antibody while nine isolates gave stronger reactions when using rabbit IgG. No reactions were observed with other plant viruses and plant proteins from healthy Datura.

Three Different Types of α-Amylases from Aspergillus awamori KT-11: Their Purifications, Properties, and Specificities.

Three forms of α-amylases, designated Amyl I, Amyl II, and Amyl III were purified to a homogenous state by several column chromatographies from a koji culture in wheat bran of a strain of black mold, which was isolated in Indonesia and identified as Aspergillus awamori. They have molecular weights of 49,000, 63,000, and 97,000 by SDS-PAGE, respectively, and the optimum pHs were 4.0 for Amyl I and 5.5 for both Amyl II and Amyl III on soluble starch. Amyl I hydrolyzed malto-tetraose, -pentaose, -hexaose, -heptaose, and ß- and γ-cyclodextrin to produce maltose and maltotriose as major products but not or hardly hydrolyzed maltose, isomaltose, maltotriose, isomaltotriose, α-cyclodextrin, or raw corn starch. On the other hand, both Amyl II and Amyl III hydrolyzed maltotriose as well as all the maltooligosaccharides described above and α-, ß-, and γ-cyclodextrin, and even raw corn starch as well as heat-gelatinized corn starch to produce maltose as a major product and glucose and maltotriose as minor products, but they did not hydrolyze maltose, isomaltose, or isomaltotriose. The limit hydrolyses of soluble starch with three kinds of enzymes were 33% for Amyl I, 35% for Amyl II, and 38% for Amyl III, the reaction products had α-anomeric forms by NMR analysis, and the blue color reaction with I2 disappeared completely at about 18% of hydrolysis of the starch for all enzymes.

Synthesis of novel oligosaccharides from leucrose by an alpha-glucosidase.

An alpha-glucosidase was purified in an electrophoretically pure state from an extract of koji culture of Aspergillus sp. KT-11. This enzyme was found to have a transferring activity when the reaction was done in a high concentration of leucrose at pH 4.5. Two kinds of transfer products, fractions I and II, were obtained from leucrose by the enzyme and they were identified as [(alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-alpha-D- glucopyranosyl-(1-->5)-D-fructopyranose] and [alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->5)-D- fructopyranose], respectively. These are considered to be novel oligosaccharides.

Molecular states of fungal nuclease composed of heterogeneous subunits as estimated from the effects of urea and chelating agents.

Fungal nuclease composed of subunits with molecular weights of 8.0 x 10(4) (80K subunit), 5.0 x 10(4) (50K subunit), and 2.5 x 10(4) (25K subunit) (K. Ito, Y. Matsuura, and N. Minamiura (1994) Arch. Biochem. Biophys. 309, 160-167) was inactivated by urea and dissociated into its subunits. The urea inactivation depended on the concentration of urea, the incubation period and the temperature. The urea-inactivated enzyme had about 25% activity restored by removal of urea, and the native form of the enzyme was also reconstituted. The urea inactivation and the dissociation of subunits were almost completely prevented by Ca2+ but not by glycerol. The enzyme was also inactivated by ethylenediaminetetraacetic acid (EDTA). From this method of inactivation, the 50K and 25K subunits were still associated, but the complex showed no nuclease activity. About 80% of the activity of the EDTA-inactivated enzyme was restored by the addition of Ca2+ or Sr2+ and 20-40% by Mn2+, Ba2+, Mg2+, or Co2+. The reactivation of the enzyme by these metal ions was accompanied by the reconstitution of the native form of the enzyme. The enzyme was inactivated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) at much higher concentrations compared with the inactivation by EDTA. On the other hand, dissociation of the subunits by EGTA proceeded in a manner similar to that of the inactivation of the enzyme by EDTA. The 50K and 25K subunits were still associated, and the complex showed nuclease activity. These results indicated that the enzyme contains two kinds of metal ions. One metal ion, represented by Ca2+, is thought to stabilize the quanternary structure of the enzyme, especially to connect the 80K subunit and the complex of the 50K and 25K subunits. Another metal ion, represented by Mg2+, is thought to be bound to the complex of the 50K and 25K subunits and to be required for activity appearance of the complex. Along with these results, possible molecular states of the enzyme under various conditions are proposed.

Evidence for an essential histidine residue on active site of human urinary DNase I: carboxymethylation and carbethoxylation.

Human urinary DNase I was inactivated by monoiodoacetate and monobromoacetate. The inactivation was greater at pH 7.2 than at 6.0 and proceeded in the presence of Ca2+. Amino acid analysis of monobromoacetate-inactivated human urinary DNase I indicated that one histidine residue per mole of the enzyme reacted with monobromoacetate. Diethylpyrocarbonate also inactivated the enzyme, which was protected by DNA in the presence of Mg2+. However, oligonucleotides did not prevent the inactivation even in the presence of Mg2+. Hydroxylamine almost completely restored the activity of the inactivated enzyme by DEP. One histidine residue per mole of the enzyme was calculated to be modified, as shown by the difference spectra of DEP-inactivated enzyme. This histidine residue seems to react with the substrate. These results provide evidence that human urinary DNase I possesses one essential histidine residue at the active site.

Purification and characterization of fungal nuclease composed of heterogeneous subunits.

A nuclease was purified from the extract of wheat bran culture of Aspergillus sp. isolated from "Katsuobushi" by a series of column chromatographies. The purified nuclease showed a single protein band on nondenaturing polyacrylamide gel electrophoresis. The enzyme showed three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their molecular weights were estimated to be 80,000, 50,000, and 25,000. The molecular weight of the nuclease was estimated to be 125,000 by gel permeation chromatography. The enzyme showed maximum activity around pH 8.0 for DNA and RNA. The enzyme required Mg2+, Mn2+, or Co2+ for the appearance of activity. The enzyme was stable until 40 degrees C and in pH range of 5-9. The stability of the enzyme for temperature increased until 50 degrees C by Ca2+. The enzyme exonucleolytically degraded DNA and RNA by 3'-->5' mode to produce 5'-mononucleotides. The fungal nuclease acted on heat-denatured DNA and native DNA and RNA, but not bis(p-nitrophenyl)phosphate, p-nitrophenyl thymidine 5'-phosphate, and p-nitrophenyl thymidine 3'-phosphate. The enzyme did not show strict base specificity for DNA and RNA, while the affinity for substrate was affected by 3'-terminal bases. The enzyme preferentially degraded poly(C) and poly(U), but hardly degraded poly(A) and poly(G). The nuclease acted on closed circular double-stranded DNA to produce open circular DNA and then linear form DNA by single-strand scission. The nicking activity was intrinsic to the enzyme.

Chemical structures of hetero-oligosaccharides produced by Arthrobacter sp. K-1 beta-fructofuranosidase.

The structures of hetero-oligosaccharides obtained by the action of transglycosylation of Arthrobacter sp. K-1 beta-fructofuranosidase, using sucrose as the fructosyl donor, and several mono- and di-sacchrides as the acceptors were investigated. The main transfer products to most reducing mono- and di-sacchrides were non-reducing oligosaccharides with a fructosyl residue linked to a hemiacetal hydroxyl group. In the presence of L-sorbose, the enzyme produced 2-O-beta-D-fructofuranosyl-alpha-L-sorbopyranoside as the major product. With D-galactose or L-arabinose, the enzyme produced not only non-reducing oligosaccharides, but also reducing oligosaccharides, which were identified as 3-O-beta-D-fructofuranosyl-D-galactopyranose and 4-O-beta-D-fructofuranosyl-L-arabinopyranose, respectively. When a non-reducing sugar such as methyl alpha-glucoside was used as an acceptor, the product formed had a fructosyl residue linked at the C6 hydroxyl group.

Human salivary endo-beta-N-acetylglucosaminidase HS specific for complex type sugar chains of glycoproteins.

The enzyme that catalyzed the conversion of human salivary alpha-amylase family A (HSA-A) to family B (HSA-B) was identified. It was partially purified from the precipitate obtained by centrifugation of human saliva at 105,000 x g for 60 min by solubilization with 3[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate and column chromatographies with Sephacryl S-300-HR and hydroxylapatite. The enzyme preparation was practically free from contaminating exoglycosidases and proteases. The enzyme cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of HSA-A, as shown by the isolation of the protein moiety which contained 1 GlcNAc and 1 Fuc residue and the sugar chain (Gal)2(Fuc)1(GlcNAc)2(Man)3(GlcNAc). This enzyme also cleaved the N,N'-diacetylchitobiose moiety of the sugar chain of human transferrin tetraglycopeptide Asn-Tyr-Asn(GlcNAc)2(Man)3(GlcNAc)2(Gal)2-Lys to yield equimolar amounts of peptide Asn-Tyr-Asn(GlcNAc)Lys and sugar chain (Gal)2(GlcNAc)2(Man)3(GlcNAc). The enzyme was identified as an endo-beta-N-acetylglucosaminidase. The enzyme acted on HSA-A with desialylated and defucosylated outer chain moieties of the sugar chains at a similar rate as that of native HSA-A. The enzyme activity was reduced to 13 and 5% using HSA-A with the sugar chains whose outer chain moieties lacked Gal and GlcNAc, respectively, from the nonreducing end. The enzyme also acted on human transferrin, calf fetuin, and asparagine oligosaccharides of transferrin and fetuin. On the other hand, the enzyme did not act on ovalbumin, RNase B, Taka-amylase, yeast invertase, and ovalbumin asparagine oligosaccharides. These results indicate that human salivary endo-beta-N-acetylglucosaminidase is specific for complex type sugar chains and can release the sugar chains from native glycoproteins and glycopeptides regardless of the existence of a Fuc residue on the proximal GlcNAc of the N,N'-diacetylchitobiose core of their sugar chains. The source of the enzyme was epithelial cells peeling from the oral cavity epithelium into saliva. The enzyme was thought to be integrated on the surface of the epithelial cell membrane. This enzyme was named endo-beta-N-acetylglucosaminidase HS. Thus, these studies indicate that the properties of the enzyme are distinct from those of known endo-beta-N-acetylglucosaminidase and endo-beta-N-acetylglucosaminidase HS is a novel endo-beta-N-acetylglucosaminidase.

Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages.

The transglycosylation reaction catalyzed by neopullulanase was analyzed. Radioactive oligosaccharides were produced when the enzyme acted on maltotriose in the presence of [U-14C]glucose. Some of the radioactive oligosaccharides had only alpha-(1----4)-glucosidic linkages, but others were suggested to have alpha-(1----6)-glucosidic linkages. The existence of alpha-(1----6)-glucosidic linkages in the products from maltotriose with neopullulanase was proven by proton NMR spectroscopy and methylation analysis. We previously reported that the one active center of neopullulanase catalyzes the hydrolysis of alpha-(1----4)- and alpha-(1----6)-glucosidic linkages (Kuriki, T., Takata, H., Okada, S., and Imanaka, T. (1991) J. Bacteriol. 173,6147-6152). These facts proved that neopullulanase catalyzed all four types of reactions: hydrolysis of alpha-(1----4)-glucosidic linkage, hydrolysis of alpha-(1----6)-glucosidic linkage, transglycosylation to form alpha-(1----4)-glucosidic linkage, and transglycosylation to form alpha-(1----6)-glucosidic linkage. The four reactions are typically catalyzed by alpha-amylase, pullulanase, cyclomaltodextrin glucanotransferase, and 1,4-alpha-D-glucan branching enzyme, respectively. These four enzymes have some structural similarities to one other, but reactions catalyzed by the enzymes are considered to be distinctive: the four reactions are individually catalyzed by each of the enzymes. The experimental results obtained from the analysis of the reaction of the neopullulanase exhibited that the four reactions can be catalyzed in the same mechanism.

Evidence for conversion of human salivary alpha-amylase family A to family B by an enzyme action.

SDS-PAGE showed that human salivary alpha-amylase family A (HSA-A) was converted to family B (HSA-B) in human saliva. This conversion did not occur in the supernatant of saliva which had been centrifuged at 105,000 x g for 60 min. An enzyme which catalyzed the conversion existed in the insoluble fraction of human saliva. The enzyme was solubilized with nonionic or zwitterionic detergents, and showed the maximum activity around pH 6. It was stable between pH 4 and 10, and at a temperature lower than 40 degrees C. The enzyme reduced the molecular weight of HSA-A (62,000) to the same molecular weight (58,000) as that of HSA-B without forming any intermediate. It also changed the PAGE pattern of multiple forms of HSA-A to the same pattern as that of HSA-B. It was not inhibited by protease inhibitors, and it did not destroy the reactivity of HSA-A with anti-human salivary alpha-amylase antiserum. The enzyme diminished the reactivity of HSA-A with concanavalin A. These results indicate that HSA-A was converted to HSA-B through the release of sugar chains by the action of the enzyme in the insoluble fraction of human saliva.

Evidence for the existence of isozymes of glucose isomerase from Streptomyces phaeochromogenes.

Glucose isomerase from Streptomyces phaeochromogenes was purified from a commercial preparation, Swetase, by DEAE-cellulose, Bio-Gel A-0.5 m, and hydroxyapatite column chromatographies. It was found to be 2 fractions; F-A, not adsorbed on hydroxyapatite and F-B, adsorbed on hydroxyapatite. They were homogeneous in ordinary and SDS-PAGE and had similarities in some enzymatic and physico-chemical properties. The differences, however, were found in the N-terminal amino acid, which was only serine for F-A while it was serine and alanine for F-B, and also in their peptide mapping patterns of digests with trypsin, Achromobacter protease I, and cyanogen bromide. The results suggest that glucose isomerase from S. phaeochromogenes was composed of the two kinds of isozymes and that each of isozymes was a tetramer constituted of non-identical subunits.

Phosphodiesterase I in human urine: purification and characterization of the enzyme.

Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.

Development of a radioimmunoassay for human deoxyribonuclease I.

A reliable radioimmunoassay (RIA) for human deoxyribonuclease I (DNase I) is described. Using delayed addition of tracer antigen, the method is sensitive (9.5 reproducible and specific. A good parallel relationship was observed between the standard curve and dilution curves for human urine and human pancreatic juice. G-actin, a naturally occurring DNase I inhibitor, caused no change in the immunoreactivity of DNase I. In healthy individuals, aged 11-90 yr, the mean serum DNase I was 18.4 ng/ml (SD 6.7 ng/ml). Increased serum DNase I occurred in patients with acute pancreatitis, renal failure, and in about one-third of patients with various malignant tumors.

Preparation of human salivary alpha-amylase specific monoclonal antibody.

A mouse hybridoma cell line which produced an anti-human salivary alpha-amylase monoclonal antibody was obtained by fusion between mouse spleen cells immunized with human salivary alpha-amylase and mouse myeloma cells, followed by screening the hybridoma cells by enzyme-linked immunosorbent assay. The hybridoma cell line (27-4-1) secreted IgG. The monoclonal antibody produced by the hybridoma showed no inhibitory effect on the activity of human salivary alpha-amylase. The specificity and reactivity of this monoclonal antibody were examined by determining the activities of human salivary and pancreatic alpha-amylases bound to the monoclonal antibody immobilized on polystyrene balls or by enzyme immunoassay with the monoclonal antibody conjugated with beta-D-galactosidase. The results revealed that the monoclonal antibody produced by the hybridoma cell line was specific for salivary alpha-amylase and absolutely unreactive to pancreatic alpha-amylase.

Human uropepsinogen is a new human pepsinogen proisozyme.

The first 20 amino acid residues of human uropepsinogen has been determined to be H2N-Ile-Met-Tyr-Lys-Val-Pro-Leu-Pro-Ile-Lys-Lys-Ile-Leu-Val Val-Pro-Leu-Ile-Val-Tyr-Met. Comparison with the sequence of human stomach pepsinogen shows that these proteins are isozymes. Comparative sequence data are presented and discussed.

Effect of dithiothreitol on activity and protein structure of human urine urokinase.

Human urine urokinase [EC 3.4.21.31] was found to be inactivated by dithiothreitol (DTT) much more severely than by 2-mercaptoethanol at the same concentration on the basis of -SH groups. Removal of DTT by dialysis restored the activities of esterase toward acetyl-glycyl-L-lysine methyl ester, plasminogen activation, and amidase toward 7-(glutaryl-glycyl-L-arginine-amido)-4-methyl coumarin. But the restoration of amidase activity was much less than that of esterase activity. The addition of DTT mediated the conversion of high molecular weight urokinase to low molecular weight urokinase, releasing several peptides. This suggests that the urokinase consists of several polypeptides linked by disulfide bonds. The molecular weight of urokinase produced with DTT was smaller than that of low molecular weight urokinase obtained by autodigestion of high molecular weight urokinase. The autodigestion was also accompanied by liberation of some peptides. But, those peptides released on autodigestion of high molecular weight urokinase were different from those appearing in the presence of DTT.

Uropepsinogen in human urine: its protein nature, activation and enzymatic properties of activated enzyme.

Uropepsinogen (UPG) in human urine was highly purified by chromatography on columns of DEAE-lignocellulose, elastin-celite, etc. The purified UPG was composed of two electrophoretically distinguishable components (UPG I and UPG II), and they were isolated in the pure state, respectively. UPG I and UPG II were the same in molecular weight (3.9 X 10(4)) and in N-terminal (leucine) and C-terminal (alanine) amino acid residues. Also, they were quite similar in amino acid composition. They were activated to uropepsin (UP I and UP II, molecular weight, 3.3 X 10(4), respectively), but the activated enzymes were the same in the various properties examined, suggesting that the difference between UPG I and UPG II is due to only a minor change in the peptide segment, perhaps by deamidation. The activation of UPG I and UPG II occurred at acid pHs, the best pH being at 2.0. Both the proenzymes previously incubated with pepstatin at pH 6.8, however, were not activated even in the following incubation at acid sides. The results obtained are discussed in regard to the origin of the proenzyme and its properties before and after activation.

Human urine DNase I: immunological identity with human pancreatic DNase I, and enzymic and proteochemical properties of the enzyme.

DNase I in human urine was purified to an electrophoretically homogeneous state by column chromatographies on DEAE-lignocellulose, hydroxyapatite, DEAE-cellulose, Sephadex G-75 and elastin-celite. The purified enzyme was immunologically identical with human pancreatic DNase I, but not with bovine pancreatic DNase I. The molecular weight and isoelectric point of the enzyme were estimated to be 4.1 X 10(4) and 3.6, respectively. The amino acid analysis revealed that 1 mol of the enzyme contained 8 mol of half-cystine. The N-terminal amino acid was identified as leucine by the dansyl chloride method. The enzyme was active in the presence of Mg2+, Co2+, or Mn2+, The optimum pH was around 6.5. The enzyme was stable in the pH range from 5.0 to 9.0 and at temperatures lower than 45 degrees C. The rate of hydrolysis of native DNA by the enzyme was twice as fast as that observed with heat-denatured DNA. This enzyme exhaustively degraded about 20% of the phosphodiester bonds in native DNA. The enzyme also degraded poly(dA) and poly(dT), but hardly degraded poly(dG) and poly(dC).