Serum Biotinidase Activity Research Articles

Human serum biotinidase is a thiol-type enzyme.

The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n = 83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1 mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005 mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis(2-nitro)benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0 mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05 mM or higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of biotinidase activity in mouse serum by inhibitors of methylation.

DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be prevented by adenine, biotin or lecithin are most probably related to the inhibition of methylation.

A sensitive radioassay for biotinidase activity: deficient activity in tissues of serum biotinidase-deficient individuals

A new, sensitive radioassay for the determination of biotinidase activity was developed which measures the release of [ 14C-carboxylj- p-aminobenzoate from N- biotinyl-[ 14 C-carboxyl]-p- aminobenzoate . Biotinidase activity in serum from normal individuals is comparable to that determined by the colorimetric assay, but the radioassay is approximately 100 times more sensitive. Biotinidase deficiency was confirmed in the serum of patients who were previously shown to have reduced activities by the colorimetric assay. Although biotinidase activity was not detectable in extracts of normal peripheral blood leukocytes and fibroblasts using the colorimetric assay, activities could be measured by the radioassay. Using this method we demonstrated deficient biotinidase activity in extracts of leukocytes and fibroblasts from affected patients.

Actinomycin D-sensitive increase in the biotinidase activity in mouse liver and serum after ethionine feeding.

Actinomycin D-sensitive increase in the biotinidase activity in mouse liver and serum after ethionine feeding.

Biotinidase activity in animal tissues.

AbstractKorvusalo, M. and J. Pispa. Biotinidase activity in animal tissues. Acta physiol. scand. 1963. 58. 13—19. — Biotinidase activity of human serum and rat, guinea pig and rabbit serum, liver and intestine was determined using biocytin as substrate. The biotinidase activities of human serum and rat serum and tissues were of the same magnitude, in guinea pig serum the activity was about 10 per cent and in rabbit serum only 1 per cent of the activity in human and rat sera. Similar although less marked differences were found in the biotinidase activity of rat, guinea pig and rabbit tissues. The biotinidase activity of human and rat sera was inhibited by p‐chloromercuribenzoate, arsenite and monoiodoacetate; it was slightly stimulated by glutathione. Ethy‐lenediamine tetraacetate and cyanide had no effect. In zone electrotrophoresis the human serum biotinidase activity had slightly smaller mobility at pH 8.6 than albumin, and moved as a single peak.