Jun Oizumi

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.

Liberation of lipoate by human serum lipoamidase from bovine heart pyruvate dehydrogenase

Lipoamidase, which hydrolyses such substrates as lipoamide, lipoylmethyl ester, lipoyllysine, and lipoyl 4-aminobenzoate (LPAB), was purified from human serum through use of synthetic substrate LPAB. The purified human serum lipoamidase showed lipoyllysine hydrolase activity (Km = 435 microM, Vmax = 64.5 nmol/min per mg of protein). The purified enzyme did not liberate the free form of lipoic acid from bovine heart pyruvate dehydrogenase (PDH). PDH was hydrolyzed quantitatively by proteinase K to lipoyllysine, which was determined by the HPLC method. Although liberation of lipoate from various lengths of lipoyl-peptides has not been tested yet, it is likely that lipoamidase requires proteinase(s) before the liberation of free lipoic acid from the enzymes.

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.

Improved high-performance liquid chromatographic determination of biotinidase activity with biotinyl-6-aminoquinoline as substrate.

An improved high-performance liquid chromatographic assay method for biotinidase activity was developed using the fluorimetric substrate biotinyl-6-aminoquinoline, which was found to be more specific than the biotinyl-4-aminobenzoate previously used. The new method measures the intensity of the fluorescent signal at wavelengths (excitation 350 nm; emission 550 nm) longer than those (excitation 276 nm; emission 340 nm) for 4-aminobenzoate. The analysis of fluorescence in the visible spectrum reduced considerably the number of interfering peaks compared with analysis in the ultraviolet region. This method also made it possible to measure the biotinidase activity directly in samples usually difficult to calculate, such as human and bovine milk or porcine serum; the use of biotinyl-6-aminoquinoline allowed the analysis of the enzyme reaction in milk and porcine serum without pretreatment or dialysis. Stoichiometric increase and decrease of the substrate and product, respectively, were demonstrated. Michaelis constants for biotinyl-6-aminoquinoline were measured at various stages of partial purification. Because the solubility of these synthetic substrates in the aqueous phosphate buffer is limited, the determination of both Michaelis constant and maximum velocity by extrapolation may be helpful for the characterization of the kinetics of biotinidase.

Neonatal pyruvate carboxylase deficiency with renal tubular acidosis and cystinuria

This report concerns a patient with severe congenital lacticacidosis associated with proximal renal tubular acidosis and cystinuria. Enzyme studies with cultured skin fibroblasts obtained from the patient revealed zero pyruvate carboxylase activity, but propionyl-CoA carboxylase activity was normal. Administration of various vitamins in large amounts did not improve the clinical condition. In contrast, the patient began to thrive when her diet was supplemented with aspartic acid, asparagine, glutamic acid, and glutamine. The particular dietary treatment used and the biochemical findings merit consideration for management of future cases.

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 in the porcine cerebrum.

Biotinidase activities found in porcine brains (n = 3) were as follows: cerebrum, 4.4 +/- 0.2 pmol/min per milligram of protein; cerebellum, 7.6 +/- 0.3 pmol/min per milligram of protein; medulla, 2.9 +/- 0.3 pmol/min per milligram of protein. These values are relatively high compared with the activities in rat or guinea pig brains. Subcellular distribution of biotinidase was found mainly in the soluble cytoplasmic fraction (S3), i.e., in the supernatant of 0.32 M sucrose S2 solution after ultracentrifugation at 105,000g for 90 min. This is in contrast to the guinea pig livers, in which the subcellular distribution of biotinidase is mainly found in the microsomal fraction. After a seven-step purification (22,200-fold enrichment), porcine brain biotinidase is identified as a single polypeptide by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and its molecular weight is determined as 68,000 Da. The isoelectric point of the enzyme was 4.3. Sialidase treatment strongly suggests the presence of sialyl residues in this enzyme. Amino acid analysis indicates relatively high hydrophilicity and high content of glycine and serine. The enzyme activity is inhibited by organic mercurials, but not by diisopropylfluorophosphate. Abundant soluble biotinidase in brain cytoplasm may play an important role which has not been discovered yet.

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.

Partial ornithine transcarbamylase deficiency associated with recurrent hyperammonemia, lethargy and depressed sensorium

A 6‐year‐old boy presented with recurrent coma associated with hyperammonemia and infection is reported. A liver biopsy revealed decreased ornithine transcarbamylase (OTC) activity (16% of normal). The enzymatic abnormality in the child is supported by the finding of elevated orotic acid excretion in his mother following a protein load, compatible with an X‐linked pattern of inheritance. Since initiation of a dietary arginine supplement, the child has not had recurrent episodes of hyperammonemia.

Determination of lipoyllysine derived from enzymes by liquid chromatography.

Lipoyllysine was liberated from the commercial enzymes bovine heart alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase. After incubation of the enzymes with pronase for 4 h, the lipoyllysine liberated was determined by high-performance liquid chromatography with ultraviolet detection at 340 nm. Standard lipoyllysine was synthesized in our laboratory. The specific determination of lipoyllysine with ultraviolet detection only at 340 nm could be utilized for the enzyme hydrolysate samples. Recoveries of lipoyllysine added (5.0 micrograms) to a reaction mixture containing protease and bovine serum albumin or ovalbumin model proteins (1.0 mg) were 116.8 and 119.5%, respectively. The lipoyllysine content in beef heart alpha-ketoglutarate dehydrogenase was 0.55 microgram/mg of enzyme and 0.83 microgram/mg of enzyme protein in beef heart pyruvate dehydrogenase.