Journal of applied biochemistry最新文献

Asparagine-linked oligosaccharides were released quantitatively by N-oligosaccharide glycopeptidase (almond) digestion from human thyroglobulins prepared from thyroid glands of normal subjects and patients with several pathological conditions. The pyridylamino derivatives of the oligosaccharides were prepared and analyzed by high-performance liquid chromatography. The content of high-mannose-type oligosaccharides was comparable to that of the complex type in normal thyroglobulins. Man9GlcNAc2 was the predominant component in the high-mannose-type region, while biantennary oligosaccharides with fucose were the major components in the complex-type region. High-mannose-type oligosaccharides were markedly decreased in thyroglobulins prepared from patients with various disorders, such as Basedow's disease, papillary carcinoma, and adenomatous goiter, whereas they were appreciably increased in thyroglobulin from diffuse goiter.

beta-Lactamase from Enterobacter cloacae P99 was immobilized onto Sepharose by the cyanogen bromide activation method and the properties of the Sepharose-bound enzyme were compared with those of soluble and cell-bound enzyme. The immobilized beta-lactamase showed enhanced stability to storage at 4 degrees C (approximately 1 year) in respect to the free enzyme in solution (few days). The optimum pH for activity is similar for both Sepharose- and cell-bound beta-lactamase and extends over a broader pH range (pH 6-9) than the soluble enzyme (pH 8-9). Immobilization leads also to significant enhancement of thermal stability. Effective enzyme inhibition by flucloxacillin occurs with both soluble and Sepharose-bound beta-lactamase, whereas the cell-bound enzyme is much less (10(-5) times) inhibited. These results indicate that immobilized beta-lactamase could be usefully employed as a tool for investigating the properties of newly designed beta-lactamase inhibitors.

Monoclonal antibodies to human luteinizing hormone (hLH) were generated by a modified hybridoma technique. Out of forty hybrid cell lines that were shown to secrete antibodies reacting with hLH, LH35 and LH40 were further characterized biochemically and immunologically. LH35 was found to secrete immunoglobulin G1 antibody specific to the alpha-subunit of LH, whereas that of LH40 was beta-subunit specific. Association constants between the antibodies and LH were determined to be 2 X 10(8) and 1 X 10(9) M-1, respectively, by using competitive radioimmunoassay and Scatchard plots. Monoclonal antibodies from LH35 and LH40 were purified from the respective ascites fluids by ammonium sulfate fractionations and DEAE ion-exchange chromatography. The purified alpha-subunit-specific antibody of LH35 was immobilized on polystyrene balls (6 mm in diameter), whereas purified LH40 antibody was conjugated with horseradish peroxidase or labeled with iodine-125. Solid-phase radio- and enzyme immunoassays were designed to measure relatively low concentrations of LH (2-100 mIU/ml). The LH surge during the midcycles of women with normal menstrual cycles could easily be detected from daily urine or serum specimens by a 1-h assay procedure. It is proposed that this new LH immunoassay procedure can be routinely used for predicting ovulation of women with normal menstrual cycles.

Teicoplanin, as well as the other antibiotics of the vancomycin group, was shown to bind specifically to D-alanyl-D-alanine-agarose (D-Ala-D-Ala-AGA) (A. Corti and G. Cassani, Appl. Biochem. Biotechnol. 11, 101-110 (1985)). This finding is extended, showing that the binding is as a function of concentration and physical form of the antibiotic in solution, i.e., monomers or micellar aggregates. At concentrations below the critical micelle concentration (CMC) teicoplanin binds with an affinity and a capacity similar to the other antibiotics of the same group such as vancomycin and ristocetin A. At concentrations above the CMC three times more teicoplanin is bound to D-Ala-D-Ala-AGA than the other two antibiotics. Equilibrium binding experiments carried out at different pHs with teicoplanin in the monomeric or micellar form indicate that the excess binding of teicoplanin occurs in the presence of micelles. Elaboration of binding data according to Scatchard indicates that the maximum binding capacity of the resin is increased 3.6 times when teicoplanin is in the micellar form. On the contrary, the apparent binding affinity is lower.

The binding mechanism of proteins to an amorphous polymer gel [formaldehyde-hydroquinone (FA-HQ)], which is a product of addition condensation of hydroquinone with formaldehyde, is examined. Proteins such as serum albumin and gamma-globulin bound to the FA-HQ gel rapidly and irreversibly (without elution by acid, alkali, urea, or detergent solution). The binding of a modified bovine albumin to the FA-HQ gel showed that the blocking of the amino groups in the albumin molecule decreased the amount of protein bound to the gel. Amino acids bound to the FA-HQ gel to a larger extent at alkaline than at neutral pH. These results supported the essential role of the amino groups in binding to the FA-HQ gel. Using the FA-HQ gel-packed glass column or polymer tube, rapid and complete removal of proteins from serum could be carried out, which suggests the usefulness of the gel for the easy pretreatment of biological samples for clinical assays using immobilized enzyme columns.

The stability of galactosyltransferase was greatly enhanced on immobilization. The immobilized enzyme was demonstrated to be insensitive to mechanical stirring for several hours, whereas its soluble counterpart was totally inactivated in less than 60 min under the same conditions. Immobilization also conferred increased thermostability to the enzyme. The Arrhenius energy of inactivation was raised from 13 kcal/mol for the soluble enzyme to 30 kcal/mol for the immobilized protein. The gel-bound enzyme could be stored at 4 degrees C for over 10 months without apparent loss of activity.

Milk xanthine oxidase oxidizes xanthine at pH 9.6 and reduces nitrates at pH 5.2. It is shown that the nitrate reductase activity requires molybdenum and sulfur-containing sites in the enzyme, whereas oxidation of xanthine also requires iron-containing sites and FAD. As the pH changes from 5.2 to 9.6, the conformation of the enzyme molecule is modified as demonstrated by changes in the absorption, fluorescence, and circular dichroism spectra. When the enzyme is treated with dithioerythritol, it may pass from the oxidase to the dehydrogenase form with a marked increase in the nitrate reductase activity.

D-Amino acid oxidase (EC 1.4.3.3) has been purified from the yeast Trigonopsis variabilis by the application of ion-exchange chromatography on DEAE-cellulose, salt precipitation, gel filtration, and hydroxyapatite adsorption. Alternatively the last two steps can be substituted by a single fast protein liquid chromatographic ion-exchange step (Mono Q). The enzyme appeared homogeneous in PAGE, but small amounts of impurities (not exceeding 5% of total protein) appeared in sodium dodecyl sulfate (SDS)-PAGE. Its Mr in SDS-PAGE is 39,000; it exhibits an isoelectric point of 4.8 and contains 7% (w/v) covalently bound carbohydrate. Its absorption spectrum is similar to hog kidney D-amino acid oxidase, indicating the presence of bound FAD, which, however, could not be separated from the enzyme under non-denaturing conditions. The enzyme is inhibited by SH-oxidizing agents, but not by metal-chelate formers and not by benzoate or toluene. It uses O2 exclusively as the only H acceptor. Km and Vmax values were determined for 15 D-amino acids, which, among 23 tested, were substrates of the enzyme. The enzyme has highest affinity for D-phenylalanine and D-leucine, but maximal activity is obtained with D-citrulline and D-isoleucine. The specific activity of the purified preparation is even higher than that of the commercially available hog kidney enzyme (21.7 vs 16 U/mg). The yeast enzyme may be a useful analytical and preparative tool in view of the difference between its substrate specificity and that of the hog enzyme.

The lactating guinea-pig mammary gland synthesizes and secretes four major milk proteins, i.e., three caseins and alpha-lactalbumin. Of these, the caseins are highly phosphorylated, a post-translational event which in the mammary gland involves a specific casein kinase, which is an integral membrane protein probably of Golgi origin. The microinjection of milk protein mRNA into Xenopus oocytes in the presence of [35S]methionine leads to the synthesis, sequestration, and secretion of proteins which coelectrophorese with alpha-lactalbumin and with partially processed caseins. That the secreted caseins were not phosphorylated was shown by the use of 32P. Either the oocytes were injected with mammary gland mRNA followed by incubation with [32P]phosphate containing media or the mRNA was co-injected with [gamma-32P]ATP and the oocytes were then incubated. In neither case were 32P-labeled caseins secreted. Golgi-rich fractions, identified by the marker enzyme galactosyltransferase, were isolated from the postnuclear supernatant of both oocytes and lactating mammary gland by sucrose density gradient fractionation. In contrast to the mammary gland fractions those derived from the oocytes contained no detectable casein kinase activity. Homogenates of oocytes do effect the phosphorylation of casein but the enzyme activity appears to be present in the soluble fraction and is not membrane bound. It is concluded that the Xenopus oocyte lacks the specific kinase that in the mammary gland phosphorylates sequestered caseins and that the phosphorylation of the caseins is not a prerequisite for their secretion by the oocyte.

Total fatty acids processed from whole brain samples removed at autopsy from men and women, 26-87 years of age, were esterified, the methyl esters analyzed by gas chromatography, and the respective profiles deduced. In the study of the effect of age and sex in relation to the fatty acid levels, regression analysis was applied to smoothened data to take care of extraneous effects or variables. Toward this end, five age groupings were formed and the regression relationships explored for each group per sex. Generally, the saturated homologs occurred in higher amounts among males of advancing age but decreased with age among females. This effect stemmed from the even-carbon acids. For the unsaturated fatty acids, the reverse behavior appeared evident. For the prominent acids, 16:0 and 18:0, the differences were moderately significant for cases 50 years and older, for 18:2, significance based on either sex was noted with all age groups.