Journal of applied biochemistry最新文献

Myeloperoxidases were purified from leukemic myeloblasts (Mbl-MPO) and from normal neutrophilic granulocytes (Gra-MPO) and were compared with each other biochemically. Gel filtration studies showed that the molecular weights (Mr) of Mbl-MPO and Gra-MPO were about 70,000 and 130,000, respectively, so that the former is almost one-half the molecular size of the latter. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 2-mercaptoethanol, both MPOs split into two major subunits, heavy and light peptide chains. However, each of these chains of Mbl-MPO seemed to be smaller than that of Gra-MPO, Mr 58,000 versus Mr 62,000 for the heavy chain and Mr 12,000 versus Mr 15,000 for the light chain. On the other hand, another band of Mr 34,000 also appeared in Mbl-MPO under nonreducing conditions and in both MPOs after being boiled. The size of this band was not different in the two MPOs, but the ratio of this band to the others was much higher in Mbl-MPO. The activity of Mbl-MPO could be demonstrated in the heavy chain of Mr 58,000, using a blotting method, and was found to be less heat stable than that of Gra-MPO. These findings indicate that Mbl-MPO may represent an incomplete, unstable half-enzyme form of Gra-MPO.

(2S,4R)-"erythro"- and (2S,4S)-"threo"-4-methylglutamic acids have been prepared by glutamate dehydrogenase-catalyzed reductive amination of 2-keto-(R,S)-4-methylglutaric acid, in the presence of NH4+ ions, NAD+, and a NADH recycling system (ethanol-alcohol dehydrogenase), followed by separation by anion-exchange chromatography. Nuclear magnetic resonance, electrophoretic, chromatographic, and optical properties of both isomers are reported, together with the conditions leading to the four-carbon epimerization.

The malolactic enzyme of Lactobacillus murinus was purified 79 fold. Mr = 220,000 as determined by gel filtration and gradient gel electrophoresis. The enzyme consists of two apparently identical subunits (Mr = 110,000) that were observed after treatment with sodium dodecyl sulfate. NAD protected the enzyme against inactivation and its addition, after dissociation, restored the malolactic activity. The apparent Km's for malate, NAD, and Mn2+ were 2.31 X 10(-2), 4.5 X 10(-4), and 1.4 X 10(-4) mM, respectively. Maximum enzymatic activity was observed at 37 degrees C and pH 5.5 in 0.2 M phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 8000 and 16,200 cal mol-1 for the temperature values more than and less than 30 degrees C, respectively. Malolactic enzyme catalyzes the NAD and manganese-dependent reaction L-malate----L-lactate + CO2. Therefore, this enzyme can be distinguished from the well-known malic enzymes [L-malate: NAD+ oxidoreductase, oxaloacetate decarboxylating (EC 1.1.1.38) or decarboxylating (EC 1.1.1.39)].

Cellulose and microcrystalline cellulose are treated consecutively with sodium periodate and urea. The interaction of urea derivatives with formaldehyde results in highly reactive groups, capable of further condensation with the amino acid residues of the proteins. The condensation of chymotrypsin, pepsin, and ovomucoid with such activated matrices has been studied in the pH interval 2 to 10. Differences have been found in the binding properties of basic and acid proteins. Satisfactory values have been obtained concerning the relative enzymatic and inhibitory activity of the immobilized products with respect to high- and low-molecular substrates. Chymotrypsin, immobilized on microcrystalline cellulose matrix, is found to manifest better catalytic properties compared with chymotrypsin immobilized on cellulose matrix. A probable sequence of the stages of chemical activation of the matrices and covalent binding of the proteins to them has been proposed. The main advantages of the proposed method consist of the high reactivity of the binding group in a wide pH range, its suitable length, and its easy synthesis.

In studies on antitumor antibody-drug conjugates as potential antitumor agents with improved tumor specificity, conjugates of mitomycin C (MMC) with the IgMs fragment of a monoclonal IgM antibody to a tumor-associated antigen (MM antigen) on mouse mammary tumor MM46 cell (anti-MM46 IgMs) were prepared by direct and bovine serum albumin (BSA)-mediated indirect conjugation. MMC was linked to the IgMs and BSA by the use of 1a-[4-(N-succinimidoxycarbonyl)butyryl]mitomycin C, which allowed the slow release of MMC. In the indirect conjugation, the thiol group of BSA was first protected as the 2-pyridyldithio group and, after the MMC binding, regenerated with dithiothreitol, and the resulting BSA-MMC was reacted with the IgMs having the maleimide group introduced with N-succinimidyl m-(N-maleimido)benzoate. Anti-MM46 IgMs-MMC was more cytotoxic against the target MM antigen-positive but Thy 1.2 antigen-negative MM46 cells than control anti-Thy 1.2 IgM-MMC. No such selective cytotoxicity was observed between anti-MM46 IgMs-MMC and anti-Thy 1.2 IgMs-MMC against the MM antigen- and Thy 1.2 antigen-negative MM48 cells. Anti-MM46 IgMs-BSA-MMC was more cytotoxic against MM46 cells than was a mixture of unconjugated anti-MM46 IgMs and BSA-MMC.

A bioluminescent assay based on the bacterial luciferase reaction has been developed for the determination of total lactate dehydrogenase and heart-specific lactate dehydrogenase isoenzyme-1 activity in serum. The lactate dehydrogenase-catalyzed reaction was measured in both directions, but NADH formation (lactate----pyruvate) is recommended because it allows the use of optimal reaction conditions. Internal calibration with a known amount of NADH accounts for possible interference from samples when both NADH formation and consumption are followed. The bioluminescent method is sensitive, has good precision, and is readily automated. Serum lactate dehydrogenase isoenzyme-1 was immunochemically isolated and the activity was assayed by bioluminescence. A good correlation between the bioluminescent assays and the conventional spectrophotometric procedure used as reference was obtained.

A series of basic proteins, rich in proline, were isolated from the salivary secretions of humans and rats. These proteins underwent metachromasia after staining with Coomassie brilliant blue R-250 in sodium dodecyl sulfate-polyacrylamide gels. The technique of destaining gels in several changes of 10% acetic acid after a 30-min staining period is a rapid method of general utility for the identification of proline-rich proteins from total cell lysates from other sources besides saliva.

The lipid composition of a human hepatoma tissue (liver cell carcinoma) was examined by thin-layer chromatography (TLC), and an unusual lipid spot was found in a large quantity. It was identified as consisting of fatty acid methyl esters by TLC and gas chromatography-mass spectrometry techniques. In the fatty acid methyl ester fraction, oleate (18:1) and palmitate (16:0) were dominant. Furthermore, very unusual fatty acid methyl esters, such as methyl 2-methyl oleate, were detected. These unusual lipids could be detected neither in cirrhotic lesions of the host liver nor in normal control tissues.

A practicable sandwich-type enzyme immunoassay for neuron-specific enolase (NSE) in human serum was established by the use of purified antibodies to bovine neuron-specific gamma gamma-enolase. The assay system consisted of polystyrene balls (6.5 mm in diameter) with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay could be performed within a working day. The assay system had a minimum detectable sensitivity of 50 pg per assay tube or 1 microgram per liter of NSE in human serum. The assay did not cross-react with nonspecific alpha alpha-enolase and muscle-specific beta beta-enolase. Coefficients of variation in within-assay and between-assay of serum NSE were less than 10%. Serum NSE values (n = 79) determined with the present method correlated well with those obtained by the previously developed method (correlation coefficient r = 0.96, y = 0.92x - 0.83). Serum NSE concentrations of healthy subjects were less than 5 micrograms per liter and those in sera of patients with small-cell carcinoma of the lung were enhanced.