Isolation and characterization of human breast milk lipoamidase.

The mean lipoamidase activity in human breast milk was found to be 0.073 nmol/min per mg (S.D. = 0.028, range = 0.020-0.123, n = 44). The mean lipoamidase activity is approximately 3-fold higher in milk than that in serum (0.023 nmol/min per mg, S.D. = 0.016, range = 0.001-0.059, n = 32). Lipoamidase was purified to 4400-fold by a four-step procedure from 330 ml of human breast milk. The purified enzyme was identified as a single band (Mr = 135,000) by sodium dodecyl sulfate/polyacrylamide electrophoresis. Analysis by Edman degradation indicated that the N-terminal amino acid was glycine. These results strongly suggest that milk lipoamidase is composed of a single polypeptide chain. The enzyme is considered to be a glycoprotein since it reacted positively to periodate-Schiff (PAS) staining. The isoelectric point of the enzyme was 4.2. After treatment of lipoamidase with sialidase, its position on isoelectric focusing gel moved from pH 4.2 to 4.6. This is strongly indicative that lipoamidase contains sialic acid residues. The optimum pH for the enzyme activity is 7.0. The Michaelis constant (KM) for lipoyl p-aminobenzoate is calculated as 25 microM. The enzyme activity was completely lost by heating 60 degrees C for 5 min. The effects of thiol-reactive agents, such as 2-mercaptoethanol (ME) and p-chloromercuribenzoate, were not significant. However, the enzyme activity was completely inhibited by 50 microM diisopropylfluorophosphate. Thus, this enzyme seemed to contain an essential serine residue in the active site.

Enkephalin hydrolysis by human serum biotinidase.

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.

Biotinidase and lipoamidase in guinea pig livers.

Guinea pig biotinidase and lipoamidase were mostly located in the liver microsomal fraction. Approx. 80% of the total activities of both enzymes were associated with the membranes subfractions of liver. The subunit molecular masses of these enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomal biotinidase and lipoamidase were 70 and 60 kDa, respectively. Sephadex G-200 gel-permeation chromatography in the presence of 0.1% Nonidet P-40 indicated that the native-state molecular masses of microsomal biotinidase and lipoamidase were 68 and 120 kDa, respectively. The isoelectric points of microsomal biotinidase and lipoamidase were 6.3 and 5.7, respectively. Linear sucrose density centrifugation analysis indicated that both enzymes exist in the rough endoplasmic reticulum. Comparison of amino acid analyses indicated a higher content of leucine and lower content of serine in lipoamidase than in biotinidase. Microsomal biotinidase and lipoamidase were classified as being thiol-type and serine-type amidases, respectively.

Biotinidase in human breast milk.

Biotinidase activity in 19 samples of human breast milk was investigated with the sensitive high-performance liquid chromatography (HPLC)-fluorometric method that we developed. All samples exhibited biotinidase activity. For mature milk the mean activity of 17 samples was 0.208 nmol.min-1.mL-1 milk (range, 0.087-0.516 nmol.min-1.mL-1) and mean specific activity was 7.51 pmol.min-1.mg-1 protein (range, 2.17-17.2 pmol.min-1.mg-1). These values are relatively low compared with the activity in human serum (5.26 +/- 2.92 nmol.min-1.mL-1 serum and 95.6 +/- 53.1 pmol.min-1.mg-1 protein; n = 246). Biotinidase activities of milk obtained at various times after birth were not significantly different. However, biotinidase activity in colostrum was about five times higher than that of mature milk. The existence of biotinidase activity in all specimens suggests that this enzyme plays an important nutritional role during infancy.

Comparative study on human milk and serum biotinidase.

Biotinidase was purified from human breast milk (4,000-fold), and was compared with human serum biotinidase (enriched 30,000-fold). The molecular weight of milk enzyme was 68,000 Da as determined by SDS-PAGE. It was definitely smaller than that of serum biotinidase (Mr = 76,000). Isoelectric point of milk biotinidase was 4.6, whereas that of serum biotinidase was 4.3. Sialic acid content in milk biotinidase was less than that found in serum enzyme. N-Acetyl-galactosamine was present in milk enzyme, whereas it was absent in serum enzyme. Milk biotinidase is O-glycosylated, whereas serum biotinidase is N-glycosylated. These differences in glycosylation suggest the existence of different types of excretion mechanisms between milk and serum biotinidase. Both biotinidases were found to be thiol-type enzyme, however, the extent of activation of the enzyme by 2-mercaptoethanol was 13-fold in milk, whilst the serum enzyme was activated only 1.5-fold. Km for biotinyl-4-amino-benzoate was 22 microM in milk enzyme and 50 microM in serum enzyme. Competitive inhibition by biotin (Ki) of milk enzyme was 43 microM and 1.3 mM for serum enzyme. These results suggest the structural differences at or near the active site of the each enzyme.

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)

The effects of biotin deficiency on organic acid metabolism: increase in propionyl coenzyme A-related organic acids in biotin-deficient rats.

Volatile organic acid levels in plasma and tissues and nonvolatile organic acid levels in urine of biotin-deficient (BD) rats were measured and compared with other factors of biotin deficiency. Biotin levels and the activities of propionyl coenzyme A (CoA) carboxylase (PCC) in the livers of these rats were decreased, respectively, to 22% +/- 3% and 3.6% +/- 0.3% of the average values of pair-fed controls. Plasma concentrations of propionate were higher (15 to 223 micrograms/mL) than those of controls (5 to 7 micrograms/mL), whereas plasma levels of 3-methylcrotonate were only minimally increased as compared with those of controls. Concentrations of these volatile acids in the tissues were similarly increased, although those in brain showed less remarkable increases as compared with levels in other tissues. In the urine of BD rats, large amounts of organic acids derived from propionyl CoA, as well as those from 3-methylcrotonyl CoA, were excreted. Plasma propionate levels were not apparently related to the severity of clinical symptoms, biotin levels, or carboxylase activities, but were related to the amounts of urinary ketone bodies, lactate, and some of the organic acids derived from branched-chain amino acids, including those from propionyl CoA.

Robertsonian translocations in Paget's disease of bone.

The karyotypes of 14 patients with Paget's disease of bone were studied. The patients were recruited from our bone metabolism clinic where they received specific therapy for their skeletal disease. Eight of the 14 patients had chromosomal translocations localized to the D and G groups. None of the patients were related to one another, nor had any had the same lifelong environment. Thus, 57% of a sample of active patients with Paget's disease had Robertsonian translocations. By comparison, an age and sex-matched group of eight controls and 13 patients with osteoporosis who had been treated with bisphosphonates demonstrated no Robertsonian translocations. The prevalence of Robertsonian translocations in 14,000 newborns was reported to be 0.1%. These data suggest that a factor from the environment introduced during the lifetime of the patient could be present and could, in addition to genetic factors, affect gene replication during the development of Paget's disease.

Biocytin-specific 110-kDa biotinidase from human serum.

Biotinidase purification from human serum was performed under new protocol. With HPLC biotinidase assay instead of colorimetric method and using non-ionic surfactant, 110-kDa biotinidase was discovered and co-purified in addition to the previously identified 76-kDa biotinidase. This newly identified enzyme accounted for 5% of the total biotinidase activity. Protein core of 110 kDa was estimated as 72 kDa by use of N-glycanase and SDS-PAGE analysis, while that of the 76-kDa enzyme was estimated as 59 kDa. Total amino acid analysis indicated 30% higher absolute amounts of amino acids in 110-kDa enzyme. The following differences were observed from kinetic study: the 110-kDa enzyme showed a 10-fold lower Km value and a 9-fold higher kcat/Km value for biocytin than those of 76-kDa enzyme. Thus, 110-kDa enzyme is more likely to be the physiological biocytin hydrolase (biocytinase), since the biocytin concentration in human serum is extremely low. The pathogenesis of an inborn error of the metabolism such as a variant form of biotinidase deficiency, which presented an atypical clinical course, might be related to these isoenzymes in terms of their different roles in the body.

Effect of plasma biotinyl-peptides on biotinidase activity.

Purified biotinidase (enriched 24,000-fold) from fresh human plasma exhibited reduced catalytic activity when incubated with heat-inactivated dialyzed plasma. The polypeptide fractions separated from the heat-inactivated dialyzed plasma using streptavidin-Sepharose resin showed the same effect on purified biotinidase. These inhibitory effects on biotinidase were partial (25-45%) rather than complete. The polypeptide fraction from streptavidin-Sepharose resin was analyzed by SDS-PAGE in the Laemmli system and by various types of HPLC. Analyses by ion-exchange and reversed-phase HPLC revealed the existence of three relatively small mol. wt polypeptides. Each of these peak fractions exhibited similar inhibitory effects on biotinidase activity. SDS-PAGE analysis indicated that the streptavidin affinity resin fraction was composed of four major polypeptides whose mol. wts were 120,000, 76,000, 53,000 and 27,000. The two bands of 120,000 and 76,000 corresponded to the mol. wts of the biotinyl subunit of pyruvate carboxylase, beta-methyl-crotonyl-CoA and/or propionyl-CoA carboxylase respectively. However, the polypeptides of mol. wts 53,000 and 27,000 were found to be two unique biotinyl-peptides present in human plasma. These bands on the gels were transblotted and exhibited a fluorescent activity after incubated with a FITC-avidin. These findings strongly suggest the existence of circulating plasma biotinyl-polypeptides as inhibitory factor(s) on human plasma biotinidase.

Interaction of metals at the reaction center of lipoamidase.

Various metals have been shown to inhibit porcine brain lipoamidase activity at 0.1 mM, but not ferrous and ferric ions. However, in the presence of ethylenediamine tetraacetic acid (EDTA) 0.1 mM iron ions did inhibit the activity. No other metals exhibited this type of increased inhibition with the addition of EDTA. The ferric- and ferrous-EDTA compounds were equally effective. Various Fe-containing compounds also inhibited the enzyme activity, the order of inhibition being: EDTA greater than o-phenanthroline greater than azide greater than citrate. Hemin also inhibited the enzyme activity strongly. However, Fe-proteins, e.g. cytochrome c, transferrin and peroxidases, were not inhibitory. These results indicate the importance of Fe ion chelates with structural and molecular size differences for interaction with the reaction center of this enzyme.

Agar plate method using Lactobacillus plantarum for biotin determination in serum and urine.

An improved agar plate method of biotin bioassay using Lactobacillus plantarum ATCC 8014 and bromocresol purple was established to determine biotin levels in human serum and urine. Samples were treated with 4.5 N H2SO4 to liberate free biotin, autoclaved for 1 h and neutralized by 4.5 N NaOH, then 10 microliters was added to wells in each plate. The biotin levels were measured in 190 serum and 59 urine samples, and the means were 2.7 +/- 0.53 ng/ml and 12.4 +/- 5.56 ng/mg of creatinine, respectively. The intra-assay coefficient varience (CV) were 3.2 (n = 20) and 1.3% (n = 23), respectively. The recovery of biotin added (10 ng/ml) to serum was 110.7%, and to urine was 99.6%. These findings suggest that this assay is sufficiently accurate and reproducible for routine use in the clinical laboratory. The excretion of orally administered biotin was also demonstrated by the method.

[Clinical evaluation of serum biotin levels and biotinidase activities in patients with various liver diseases].

In order to evaluate the clinical significance of serum biotin and biotinidase in liver disease, serum biotin levels and biotinidase activities were determined in 83 patients with various liver diseases and 10 healthy controls. Serum biotin levels and biotinidase activities were determined by a simplified lactobacillus plantarum bioassay and liquid chromatography with fluorimetric detection respectively. Serum biotin levels in decompensated liver cirrhosis, hepatoma and fulminant hepatitis were found to be significant low compared with healthy controls, while it was significant high in autoimmune hepatitis. There was no significant difference between serum biotin levels in the other liver diseases and healthy controls. In various liver diseases except for both acute hepatitis and alcoholic liver disease biotinidase activities were significantly reduced than in healthy controls. Serum biotinidase activities were correlated with serum albumin, prothrombin time, ChE and total cholesterol respectively, suggesting that biotinidase activities may reflect the degree of liver damage. These results seem that biotin deficiency may occur in some cases of severe liver diseases.

Human serum lipoamidase.

Thirty-two human serum specimens were assayed for lipoamidase (lipoyl-4-aminobenzoate hydrolase) activity. All sera had lipoamidase activities. This substrate was newly synthesized by us and had a satisfying purity as evaluated by HPLC-fluorimetric detection. Product (p-aminobenzoate) liberated was determined directly by the HPLC-fluorimetric method. Liberation of the product was linearly continued for 6 h. The pH optimum of serum lipoamidase was found to be 7.0. The effect of substrate concentration on human serum lipoamidase activity was examined and the reaction was saturated at 0.1 mmol/l. The sera obtained were from individuals aged from 1 to 8 years. The mean value of serum lipoamidase activity was found to be 1.50 U/l (SD 1.037, range 0.04-3.75, n = 32). The difference of sex effects was analyzed and no significant difference was found (males: n = 14, mean 1.48, SD 1.162, range 0.04-3.75; females: n = 18, mean 1.52, SD 0.963, range 0.48-3.51) among this age group. Biotinidase activity was also determined in these 32 serum specimens and the correlation was examined. The mean biotinidase activity was 3.16 U/l (SD 2.567, range 0.35-9.37). The correlation coefficient (r) between lipoamidase activity and biotinidase activity was 0.8931. Although the physiological significance of lipoamidase has not been known, the enzyme might play an important role in recycling of lipoate as biotinidase does.