The characteristics and applications of recombinant cholesterol dehydrogenase.

Mass production of an r-CDH derived from Nocardia species was made possible by gene technology. (Horinouchi et al., Applied and Environmental Microbiology, 57, 1386-1393 (1991)). However, the characteristics of the r-CDH have not been studied in detail and have not been improved enough for industrial use. We accordingly characterized both the native-CDH and the r-CDH prepared from Streptomyces lividans. Both CDHs were monomers with molecular masses of 37 kDa. The Km of r-CDH was 2.50 x 10(-3) M for cholesterol and 2.33 x 10(-4) M for NAD. The activators of CDHs were TritonX-100 and cholate. TritonX-405, Ag+, and Zn2+ inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37 degrees C, 60 min), while the r-CDH showed a residual activity of 87% (37 degrees C, 60 min). The r-CDH is an enzyme with high substrate specificity for cholesterol as well as native CDH and higher thermal stability than native CDH. We have developed a novel serum cholesterol assay using the r-CDH, which permits the direct measurement of cholesterol by measuring NADH reaction products. We conclude that this r-CDH enzyme is useful and can be used to measure cholesterol in a clinical chemistry setting.

Enzyme electrochemical preparation of a 3-keto derivative of 1,5-anhydro-D-glucitol using glucose-3-dehydrogenase.

A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. alpha-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion of 1,5-anhydro-D-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol (3-ketoAG), which was identified by 13C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L.h), and the electrochemical yield of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%.

Effect of growth substrates on production of new soluble glucose 3-dehydrogenase in Halomonas (Deleya) sp. alpha-15.

Halomonas (Deleya) sp. alpha-15 produces new co-factor binding soluble glucose 3-dehydrogenase (G3DH), which oxidizes the third hydroxy group of pyranose. This study investigated the condition of efficient production of G3DH using Halomonas (Deleya) sp. alpha-15. This enzyme was inducible, and alpha-methyl-D-glucoside, isopropyl-thiogalactopyranoside (IPTG) and lactose were revealed to be suitable carbon sources for G3DH induction. Maximum G3DH production was achieved by using minimal medium containing 0.8% (w/v) lactose with a productivity of 470U/l.

A new enzymatic method for the determination of inulin.

A new enzymatic method for the determination of inulin in plasma and urine, using inulase (EC 3.2.1.7), fructokinase (EC 2.7.1.4), phosphoglucoisomerase (EC 5.3.1.9) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) is described. The assay is based on the hydrolysis of inulin or Inutest (INutest which is the injectable form of inulin), by inulase and the determination of fructose released. The assay was linear up to 2 g/L of Inutest. The within-batch and between batch coefficients of variation were 2.3% and 2.2%, respectively. Recovery of added Inutest from plasma and urine was 98-102%. There was no interference from glucose (27.7 mmol/L), fructose (1.7 mmol/L) or mannose (1.7 mmol/L). When inulin clearance (using this method) and thiosulphate clearance were compared in 37 patients the inulin clearance was 9.3 mL/min (12%) lower than the thiosulphate clearance. We conclude that this enzymatic method is a simple and specific method.

Isolation and characterization of a serine proteinase, inactivating m-subunit of lactate dehydrogenase, from Penicillium citrinum KE-1.

A selective inactivating enzyme for the m-subunit of lactate dehydrogenase (LDH) was found in the culture filtrate of Penicillium citrinum KE-1, newly isolated from soil. The enzyme was purified from the culture filtrate by ammonium sulfate fractionation, column chromatography on CM-Sepharose CL-6B, and gel filtration on Sephadex G-100. The purification was 124-fold with an activity yield of 81%. The purified enzyme gave a single band, corresponding to a molecular weight of 32,000, on SDS polyacrylamide gel electrophoresis, and the isoelectric point was 9.5. The enzyme specifically inactivated the m-subunit of LDH but showed no activity on the h-subunit of LDH. The enzyme, named KE-1 proteinase, proved to be a serine-type proteinase. Limited proteolysis of native m-subunit of LDH was assumed to result in a loss of enzyme activity.

Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase.

We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at Ileu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run < 4.45%) and a coefficient of correlation of r = 0.985 (N = 125).

Specific assay of serum lactate dehydrogenase isoenzyme 1 by proteolysis with alpha-chymotrypsin and protein denaturation.

We devised a method for assaying serum lactate dehydrogenase isoenzyme 1 (LD-1) activity specifically by preincubation with alpha-chymotrypsin and guanidine. Cleavage of phenylalanine bonds in the loop of A and B subunits of LD-3, LD-4, and LD-5 isoenzymes (residues 117-119) by incubation with alpha-chymotrypsin for a short time completely inactivated these isoenzymes and partially inactivated LD-2. Addition of guanidine (0.50 mol/L, pH 7.8) to the incubation mixture containing the chymotrypsin completed the inactivation of LD-2. As much as 4000 U/L of LD-2, LD-3, LD-4, and LD-5 were inactivated, whereas LD-1 was affected only slightly. Results by this method (y) correlated well with those by the Roche Isomune immunochemical LD-1 method (x): y = 0.98 x -0.11, r = 0.99 (n = 60). Within-run CVs were 0.5-2.5%. Several common interferents had no effect. In 500 healthy people, serum LD-1 ranged between 66 and 130 U/L, with a mean +/- SD of 88 +/- 15 U/L.

New automated measurement of mitochondrial aspartate aminotransferase with use of protease 401.

Total mitochondrial aspartate aminotransferase (EC 2.6.1.1), the sum of apo- and holo-mitochondrial aspartate aminotransferase activity in human serum, was measured by using a proteolytic method: inactivation of cytosolic aspartate aminotransferase with cytosolic aspartate aminotransferase-inactivating protease 401 from Streptomyces violaceochromogenes. Cytosolic aspartate aminotransferase is completely inactivated, and apo-mitochondrial aspartate aminotransferase is completely activated by pyridoxal 5'-phosphate within 5 min. Results by the proposed method correlated well with those by an immunochemical method (r = 0.994, n = 145) and showed excellent inhibitory activity of the protease for holo- and apo-cytosolic aspartate aminotransferase up to 5000 U/L and activation of mitochondrial apo-aspartate aminotransferase up to 2000 U/L in the presence of 100 mumol of pyridoxal 5'-phosphate per liter. Within-run Cvs were good (1.13-7.49%). Mean values for total mitochondrial aspartate aminotransferase and apo-mitochondrial aspartate aminotransferase activities in serum of the healthy subjects were 4.8 (SD 0.9) and 1.8 (SD 0.8) U/L, respectively (n = 154). Various common interferents tested did not affect this assay.

Proteolytic measurement of mitochondrial aspartate aminotransferase in human serum.

A new proteolytic measurement of serum mitochondrial aspartate aminotransferase was evaluated using cytosolic aspartate aminotransferase inactivating protease. Some of the proteases, such as, alpha-chymotrypsin, subtilisin and cytosolic aspartate aminotransferase inactivating protease 401 from Streptomyces species, also specifically inactivated cytosolic aspartate aminotransferase, but not mitochondrial, aspartate aminotransferase. The protease 401 was the most heat stable for storage and showed a higher inactivation rate for cytosolic aspartate aminotransferase--up to 7000 IU/L--more than 200-fold the upper limit. The coefficient of variation of the proteolytic method was less than 10%. Results by the present method correlated with those by the immunochemical method (r = 0.970) and the regression curve was Y = 0.95X + 1.60 (Y: immunochemical method; X: proteolytic method). In the present assay system, reference values for mitochondrial aspartate aminotransferase activity in 500 healthy people ranged from 2.0-7.2 U/L (mean 3.8 U/L).

Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase.

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.