Characterization of human tissue carnosinase.

Human tissue carnosinase (EC 3.4.13.3) had optimum activity at pH9.5 and was a cysteine peptidase, being activated by dithiothreitol and inhibited by p-hydroxymercuribenzoate. By optimizing assay conditions, the activity per g of tissue was increased 10-fold compared with values in the literature. The enzyme was present in every human tissue assayed and was entirely different from serum carnosinase. Highly purified tissue carnosinase had a broader specificity than hog kidney carnosinase. Although tissue carnosinase was very strongly inhibited by bestatin, it did not hydrolyse tripeptides, and thus appears to be a dipeptidase rather than an aminopeptidase. It had a relative molecular mass of 90 000, an isoelectric point of 5.6, and a Km value of 10 mM-carnosine. Two forms of kidney and brain carnosinase were separated by high-resolution anion-exchange chromatography, although only one form was detected by various electrophoretic methods. Homocarnosinase and Mn2+-independent carnosinase were not detected in human tissues, although these enzymes are present in rat and hog kidney.     

Separation and characterization of two carnosine-splitting cytosolic dipeptidases from hog kidney (carnosinase and non-specific dipeptidase)

High performance anion-exchange chromatography was used to separate two carnosine-hydrolysing dipeptidases from hog kidney. Both enzymes (peaks I and II) were cytosolic and were activated and stabilized by Mn2+ and dithiothreitol. Peak I had a narrow specificity when assayed without added metal ions, but a broad specificity in the presence of Mn2+ or Co2+. Peak II was inactive unless both Mn2+ and dithiothreitol were present. Bestatin and leucine inhibited peak II, but not peak I. Peak I had a Km of 0.4 mM carnosine, a pI of 5.5 and a Mr of 57,000. Peak II had a Km of 5 mM carnosine, a pI of 5.0 and a Mr of 70,000. Hog and rat brain and liver carnosinase activity was completely inhibited by bestatin, indicating that these organs contained peak II, with little or no peak I enzyme. Hog kidney peak I contained the classical carnosinase of Hanson and Smith, who first described this enzyme. It also contained activity against homocarnosine ("homocarnosinase") and showed "manganese-independent carnosinase" activity. These three activities could not be separated using 8 different chromatographic procedures; it was concluded that they are attributable to one enzyme. It is recommended that the name carnosinase be retained for this enzyme and the names "homocarnosinase" and "manganese-independent carnosinase" be withdrawn. The properties of hog kidney peak II closely resembled those of human tissue carnosinase (also known as prolinase, a non-specific dipeptidase), mouse "manganese-dependent carnosinase" and a rat brain enzyme termed "beta-Ala-Arg hydrolase". Since these terms appear to represent closely related enzymes with broad specificity, the recommended name for each is "non-specific cytosolic dipeptidase".     

Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase.

1. The brain and ocular fluid of skipjack tuna (Katsuwonus pelamis) contained high levels of N-acetylhistidine deacetylase. 2. This enzyme had a molecular weight of about 120,000 and was activated by zinc or cobaltous ions. 3. Cod (Gadus callarias) brain, ocular fluid and muscle contained a similar metal-activated thiol hydrolase, the muscle enzyme being known as anserinase. 4. The purified enzymes hydrolyzed N-acetylhistidine, carnosine, homocarnosine, anserine and certain other dipeptides. 5. Their specificity resembled that of hog kidney homocarnosinase. 6. In both fish, brain and ocular fluid were rich sources of this hydrolase, whereas muscle contained only trace amounts.     

Isolation and Properties of Dextranases from Bacteroides oralis Ig4a

Extracellular dextranases were extracted from a dextran-degrading microorganism, Bacteroides oralis Ig4a, which had been isolated from human dental plaque, and purified. Crude enzyme preparations obtained from a broth culture supernatant by salting out with ammonium sulfate were subjected to column chromatography on DEAE-cellulose and subsequent Bio-Gel p-100, followed by isoelectric focusing. Two kinds of enzyme preparations, Enzymes I and II, with the ability to degrade soluble dextran were obtained. The optimal pHs of Enzymes I and II were 5.5 and 6.8, and the isoelectric points were pH 4.5 and 6.5, respectively. The molecular weights of Enzymes I and II were estimated by SDS-PAGE to be 44,000 and 52,000. Both enzymes were inhibited by Pb²⁺ and Fe³⁺, but not by Ca²⁺, Mg²+, Zn²⁺, or Fe²⁺. Neither the presence of EDTA nor iodoacetamide had any appreciable effect on the enzyme activity. The enzyme activity was independent of any of these metal ions. Enzyme I liberated glucose, isomaltose, maltotriose and higher oligosaccharides from dextran. In contrast, Enzyme II liberated only glucose from dextran and was assumed to be an exoglycosidase. Neither of the enzymes degraded modified insoluble glucan, which is a partially oxidized mutan of S. mutans containing predominantly α-(1, 3) linkages.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

N-acetyl-aspartate amidohydrolase: purification and properties.

Abstract— The enzyme in rat brain responsible for the de-acetylation of N-acetyl-aspartic acid has been partially purified. In contrast to the enzyme from hog kidney which is stable at 70°C, it rapidly denatures above 57°C. The rat brain enzyme has the same K_m for its substrate and the same solubility in ammonium sulphate solution as the hog kidney enzyme. Results of migration on starch gel electro-phoreses and isoelectric focusing indicate a pI for the amidohydrolase of 5.1. A variety of potential substrates, modulators, and inhibitors have been examined.     

Purification and characterization of an aminopeptidase fromStreptococcus mitis ATCC 903

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract ofStreptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-ß-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-ß-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0–7.2 and at 37–40°C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co²⁺ ions. Hg²⁺ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co²⁺ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co²⁺ ions.     

REGIONAL DISTRIBUTION OF HOMOCARNOSINE, HOMOCARNOSINE-CARNOSINE SYNTHETASE AND HOMOCARNOSINASE IN HUMAN BRAIN

Homocarnosine (HCarn) content varied over a 6-fold range in different regions of autopsied human brain, being highest in the dentate nucleus and the inferior olive, and lowest in the caudate nucleus and mesolimbic system. HCarn content was similar in biopsied and autopsied frontal cortex. Very little if any carnosine (Carn) was present in human brain, except for the olfactory bulb, where Carn may have comprised 20% of the imidazole dipeptides present. Only HCarn was present in human CSF. HCarn-Carn synthetase enzyme activity in biopsy specimens of human frontal and temporal cortex was approx 10 times greater than has been reported for rat cerebral cortex. The enzyme synthesized Carn 3–5 times as rapidly as HCarn, when β-alanine (β-Ala) or GABA substrate concentrations were 10 MM. The synthetase was found to have an apparent K_m of 1.8 mM for β-Ala, and 8.8 mM for GABA. HCarn-Carn synthetase activity decreases rapidly after brain death, and was not detectable in autopsied brain specimens frozen more than 6 h after patients’deaths. Homocarnosinase activity was determined in brain, using L-[γaminobutyryl-1-¹⁴C]HCarn as substrate, and measuring radioactive GABA produced by hydrolysis of HCarn at pH 7.2 in the presence of Co²⁺ ions. Homocarnosinase activity was similar in biopsied and autopsied human cerebral cortex, and appeared to be stable for at least 10 h after death in unfrozen brain. Differences in the regional distribution of HCarn-Carn synthetase and homocarnosinase activities, as well as regional differences in GABA content in human brain, do not readily account for regional differences in HCarn content, nor do they suggest a physiological role for HCarn.     

Isopentenyl pyrophosphate isomerase from pig liver

Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) from pig liver has been purified 197-fold. The preparation was estimated to contain less than 10% of contaminating protein. The molecular weight determined by gel filtration was 82,500 ± 3,000 and the isoelectric point from isoelectric focusing was in the range 6.0–6.2. N-terminal analysis showed the presence of both leucine and proline. The pH optimum of the enzyme preparation was 6.3. After dialysis against EDTA, activity was restored by either Mn²⁺ or Mg²⁺, the former being more effective. At the optimum pH and concentration of Mn²⁺, K_m and V were 2.7 μm and 6.7 μmol min^?1 mg^?1, respectively. The enzyme was partially inhibited by a variety of terpene mono- and pyrophosphate esters, by inorganic phosphate ions, and by acetate ions; essentially complete inhibition by sulfhydryl-blocking reagents was observed. ATP partially inhibited, the degree of inhibition showing a sigmoid dependence on ATP concentration. Monothiols and dithiothreitol activated the enzyme, as did mevalonic acid.     

Properties of New Enzyme Glycerol Oxidase from Aspergillus japonicus AT 008

Purified glycerol oxidase from Aspergillus japonicus AT 008 was homogeneous by ultracentrifugation and acrylamide gel electrophoresis. The molecular weight was determined to be 400,000 by sedimentation equilibrium, and the isoelectric point was found to be 4.9 by isoelectric focusing. The enzyme showed spectral characteristics of a heme protein. The reduced form possessed absorption maxima at 557 and 430 nm and the oxidized one at 557, 530, 420, 280, and 238 nm. The heme in the enzyme was identified as protoheme IX (one mol per mol of enzyme protein).

Glycerol was the best substrate for the enzyme, and the Km value for glycerol was determined to be 10.4 mm. Dihydroxyacetone was oxidized at 59% of that for glycerol, but glycerol 3-phosphate, dihydroxyacetone phosphate, methanol, and ethanol were not oxidized at all. The enzyme had an optimal pH at 7.0 with glycerol as substrate, and the enzymatic activity increased by treatment in alkaline pH. The enzyme was also activated by addition of several divalent metal ions including Zn²⁺, Ni²⁺, and Mg²⁺.     

Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67

Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoelectric point at pH 9.75. The results of amino acid composition analyses showed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °C respectively. The enzyme was significantly activated by the addition of 1 mM Mn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺ ions in the reaction mixture, and its thermal stability was significantly increased by Ca²⁺ ion. Received: 31 August 1998 / Received last revision: 1 December 1998 / Accepted: 20 December 1998     

Purification and Properties of a Chromobacterium Lipase with a High Molecular Weight

A lipase with a high molecular weight was purified from Chromobacterium viscosum by chromatography using the Amberlite CG–50 and Sephadex G–75. The purified lipase (Lipase A) was found to be homogeneous by disc electrophoresis.

Lipase A had an optimum pH around 7 for lipolysis of olive oil and the enzyme was stable at the range of pH 4 to 9 and below 50°C. Zn²⁺, Cu²⁺, Fe³⁺ and high concentrations of l-cysteine, iodoacetic acid and NBS had remarkable inhibitory effects. Bile salts were activator. Lipase A was more active on water insoluble esters than water soluble esters. The isoelectric point of the enzyme was pH 4.7.     

Purification, enzymatic properties of a recombinant d-hydantoinase and its dissociation by zinc ion

Summary A d-hydantoinase was expressed in the soluble form by a recombinant E. coli strain, pE-HDT/E. coli BL21 in LB medium. The enzymatic activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm. The expressed enzyme was efficiently purified by three steps, ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography and Sephacryl S-200 size-exclusion chromatography. With the above purification process, the enzyme was purified to more than 95% purity as estimated by SDS-PAGE. The overall recovery of enzymatic activity was 54.4% and the specific activity for substrate dl-hydantoin achieved 48 U/mg. The purified enzyme appeared as a dimer with a molecular mass of 103 kDa, as measured by size-exclusion chromatography. The enzyme was stable from pH 6 to 12 with an optimum pH at 9.5 The optimum temperature of the enzyme was 45 °C and it activity was rapidly lost over 55 °C. Divalent metal ions, including Co²⁺, Mn²⁺ and Ni ²⁺ ions obviously enhanced the enzymatic activity, while Zn²⁺ ion had a slight inhibitory effect. In addition, the dissociation of purified enzyme into its subunits occurred in the presence of 1 mM Zn²⁺ ion. The effect of different metal ions on the d-hydantoinase activation/attenuation was discussed.     

The isolation and characterization of copper-resistant mutants ofAspergillus nidulans

The presence of aminopeptidases in the cytoplasm, in the cell wall, and in the cytoplasmic membrane fractions ofStreptococcus sanguis 903 was demonstrated by isoelectric focusing in combination with enzyme-staining procedures. The cytoplasm and the cell wall both had two aminopeptidases (pI 4.25 and 4.3) with broad substrate specificities and one enzyme (pI 4.2) specific for arginine substrates. The former enzymes were both stimulated by Co²⁺ ions; the latter enzyme had no metal cofactor. The cytoplasmic membrane aminopeptidase (pI 4.65) was arginine specific and was not stimulated by metal ions.     

Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from Chloroflexus aurantiacus

A maltotetraose- and maltotriose-producing amylase which is stable at alkaline pHs and high temperatures was detected in the culture filtrate of a strain of Chloroflexus aurantiacus J-10-F1, a thermophilic, green, photosynthetic bacterium. The enzyme was purified to homogeneity, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by means of ultrafiltration, ammonium sulfate fractionation, and DEAE-cellulose, hydroxyapatite, and high-performance liquid chromatographies. The molecular mass of the purified enzyme was estimated to be about 210,000 Da. The isoelectric point of the enzyme was estimated to be 6.24 by polyacrylamide gel electrofocusing. The amylase was stable up to 55°C and at alkaline pHs of up to 12.0. The optimum pH and temperature of the enzyme activity were 7.5 and 71°C, respectively. Metal ions such as Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, and Ni²⁺ strongly inhibited the enzyme activity. The enzyme activity was reactivated specifically by Ca²⁺ after the enzyme was treated with 1 mM EDTA. This enzyme could digest various kinds of raw-starch granules from corn, cassava, and potato. Both maltotetraose and maltotriose were formed as the main enzymatic products from soluble starch.     

Purification and Characterization of a Fibrinolytic Protease from a Culture Supernatant of Flammulina velutipes Mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS–PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting α-chain over β-and γ–γ chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 °C. The protease activity was inhibited by Cu²⁺, Fe²⁺ and Fe³⁺ ions, but was found to be enhanced by Mn²⁺ and Mg²⁺ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.     

Characterization of membrane-associated arginine aminopeptidase inStreptococcus sanguis 903

Extracts of cytoplasmic membranes ofStreptococcus sanguis 903 were analyzed for aminopeptidase activity by isoelectric focusing in polyacrylamide gel and enzyme staining with 16 different aminopeptidase substrates. A single aminopeptidase with specificity for aminoterminal arginine was detected. The enzyme was stimulated by dithiothreitol and-mercaptoethanol. Urea, sodium dodecyl sulfate (SDS), andp-chloromercuribenzoate were inhibitory. Metal ions had little or no effect on activity, except that Hg²⁺, Cu²⁺, and Ni²⁺ were inhibitory. The pH optimum for activity was at 7.2. The molecular mass estimated by SDS-polyacrylamide gel electrophoresis was 170 kDa.     

Purification and Properties of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI = 9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5 ~ 10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41,000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA > native DNA DNA ≧ RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15 min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercapto-ethanol and glutathione had no effect. Polyamìnes showed no activation of the nuclease for DNA degradation.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.