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A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.

Human erythrocyte Mn(2+)-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn(2+)-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'.

The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50 mm sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35% and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (−15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.

Chymotrypsin and trypsin inhibitors were produced simultaneously in the culture broth of Streptomyces libani S–35. These inhibitors were purified and obtained as white amorphous powders. The properties of the chymotrypsin inhibitor were similar to those of known chymostatins except that it was positive for Sakaguchi reaction. The presence of arginine residue was further confirmed by 13C-NMR study and, therefore, the inhibitor was presumed to be a new Sakaguchi-positive chymostatin in which the capreomycidine of chymostatin B was replaced by arginine. The properties of the trypsin inhibitor were the same as those of antipain. The Sakaguchi-positive chymostatin and the antipain were the same in amino acid sequence except that the former had phenylalaninal and the later had argininal at the C-terminal.

Acid carboxypeptidase of Penicillium janthinellum IFO–8070 was produced effectively in submerged culture on a medium of 4 ~ 5% rice bran. The enzyme production was enlarged to volume cultivation of 150-liters in a 200-liters jar fermentor, and the yield of acid carboxypeptidase per milliliter filtrate reached to the maximum 3 days after inoculation.Acid carboxypeptidase of low molecular weight (M.W. = 51,000) produced in the liquid culture broth was purified and crystallized in a large scale. Purification steps include Amberlite CG–50 treatment, ammonium sulfate precipitation, dialysis using “Diaflow,” activated charcoal treatment, and condensation using collodion-bag, or condensation and dialysis using “Diaflow.”The crystals of the acid carboxypeptidase suspended in 50 mm acetate buffer (pH 3.7) were completely stable for six months at 5°C. On the other hand, at low enzyme concentration (0.01 U/ml) in 50 mm acetate buffer (pH 3.7), crystallized enzyme was somewhat labile, whereas, this inactivation was c...

In a previous paper it has been described that α-amylase formation in Aspergillus oryzae is stimulated by soluble starch, glycogen and maltose, whereas it is inhibited by glucose, which is added into a growing medium or a secondary incubation medium as the carbon source. The present paper reports that isomaltose and panose are the most effective inducers among a large number of sugars examined here, and suggests the importance of transglucosidase action demonstrated in view of α-amylase formation. The initial action of inducers in this system is also discussed.

α-Amylase in Aspergillus oryzae is generally known to be present in mycelium as well as in medium, whether it is grown with or without shaking. The interesting fact is reported in this paper, when A. oryzae was grown in a phosphate deficient medium, however, α-amylase accumulation occurred only within the cell. From the results of pH-activity curve, starch gel zone electrophoresis and differential centrifugation, there seemed to be no distinctions between exo- and endocellular α-amylase. The liberation of α-amylase from the cell was observed to be dependent on pH and the concentrations of anions. The significance of the α-amylase accumulation and liberation in this system is discussed.

α-Galactosidase was isolated from the culture broth of Streptomyces olivaceus and was partially purified by chromatography on a DEAE-sephadex column. The optimum pH of the preparation was found to be 5.2 for raffinose and the preparation was inactivated completely by maintaining it at 60°C for 15 minutes. p-Chloromercuribenzoate, HgCl2 and AgNO3 caused complete inhibition of the enzyme activity at 2 × 10‒5 M concentration. The preparation showed transglycosylase activity. A sugar spot, chromatographically identical with that of stachyose, appeared in the digest of raffinose. However, the preparation hydrolyzed raffinose completely into galactose and sucrose after a prolonged incubation.A simple raffinose estimation method was developed using the enzyme preparation, and it was found that the method allowed to estimate 125~500 μg of raffinose with an accuracy of ±5%. The method was applied to the estimation of raffinose in beet molasses.

It was previously reported that α-amylase accumulation is caused within the mycelium grown in a phosphate deficient medium and the concentration of anions or pH in a surrounding medium is responsible for its liberation. As it was subsequently found that α-amylase liberation from the mycelium of Aspergillus oryzae is stimulated by peptone, an attempt was made on purification of effective substances from it. The present paper describes on purification and properties of phosphopeptides found as an effective substance for α-amylase liberation, and discusses on the stimulation effect, comparing with the effects on pH and concentration of anions which were previously observed.

In the previous paper, it was reported that the higher molecular weight luteose which consisted of β-1,6-linked d-glucose units was made by Pen. aculeatutn var. apiculatum. The isolation of the microorganism which is able to produce β-d-1,6-glucanase and some properties of this glucanase were described in this paper. The optimum reaction temperature for this enzyme was at about 40°C, and the optimum pH was at about 6.7. At the maximum degree of the hydrolysis, only d-glucose and gentiobiose were detected, and at this point, the hydrolysis degree of higher molecular weight luteose was about 55% as d-glucose. The formation of β-d-1,6-glucanase was induced by the addition of higher molecular weight luteose. From the property of this enzyme, the authors proposed the name “lutease” for this enzyme.

An enzyme, which catalyzes the isomerization of D-glucose to D-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only D-glucose but also D-mannose to D-fructose, and NAD and Mg++ are required as cofactor for this isomerization. The properties of this enzyme were sum-marized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg++, Mn++ or Co++. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu++, however, the activity was recovered by adding cysteine or glutathione.

It has been found that exocellular α-amylase could be fixed to mycelia of Aspergillus oryzae at an acidic pH region and fixed α-amylase was released reversely at an alkaline pH region. The fixation has more remarkably been observed in mycelia obtained from a phosphate deficient medium where endocellular accumulation of a-amylase occurs more easily than in an ordinary mycelia which secrete a large amount of α-amylase into medium. Bound form of α-amylase was more resistant to low pH and less active than the free form. The results appear to support the previous suggestion that a large quantity of endocellular α-amylase might be located on mycelial surface of the mold.

A newly isolated bacterium, strain S-48, having mannose isomerase which converts d-mannose to d-fructose was identified as Xanthomonas rubrilineans. From the results of the investigation on the isomerase activities of bacteria belonging to Xanthomonas examined, it was found that the isomerase was found in all species of Xanthomonas examined. The isomerase was produced not only when the bacteria were grown on mannose, but also when on glucose, fructose, galactose, xylose, sucrose or starch, and even on bouillon medium without carbohydrate. Therefore, the formation of mannose isomerase of these bacteria seemed to be constitutive.

A newly isolated bacterium, strain S-48, having mannose isomerase which converts d-mannose to d-fructose was identified as Xanthomonas rubrilineans. From the results of the investigation on the isomerase activities of bacteria belonging to Xanthomonas examined, it was found that the isomerase was found in all species of Xanthomonas examined. The isomerase was produced not only when the bacteria were grown on mannose, but also when on glucose, fructose, galactose, xylose, sucrose or starch, and even on bouillon medium without carbohydrate. Therefore, the formation of mannose isomerase of these bacteria seemed to be constitutive.

Monofluoroacetate (FA) is well known as a toxic substance and has been used as an insecticide: C-F linkage of which is very stable. Bacteria, capable of being grown in a defined salt medium containing FA as a sole source of carbon, have been isolated from soil. It has been found that the defluorination of FA involved an enzyme which catalyzes the conversion of FA to glycolate and fluoride ions and that the enzyme was induced when bacteria were grown in a defined salt medium containing FA as a sole source of carbon. The activity of defluorination was fairly stable and strong in this organism.