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1.
Anal Biochem ; 199(1): 18-24, 1991 Nov 15.
Article in English | MEDLINE | ID: mdl-1807157

ABSTRACT

Pyridoxamine 5'-phosphate in 18 microliters of human capillary blood plasma is determined by catalytic amplification using the apoenzyme of aspartate aminotransferase. Prior isolation from interfering substances is accomplished by employment of a cation exchange resin in batch operation. The procedure consists of the following stages. Stage I, denaturation of proteins. Trichloroacetic acid is used to precipitate plasma proteins and liberate any bound coenzyme. Dilute NaCl is added to expand the volume thus minimizing coenzyme entrapment in the precipitate. Stage II, isolation of the coenzyme. A sulfonated polystyrene ion exchange resin is used inside a centrifugal filter. Pyridoxamine 5'-phosphate in the supernatant from Stage I adsorbs to the resin. Pyridoxal 5'-phosphate, other organic phosphates, and Pi are removed by centrifugation. Rinsing with dilute NaBH4 destroys traces of pyridoxal 5'-phosphate and washes off residual inhibitors. Pyridoxamine 5'-phosphate is then desorbed with NaOH and Tris buffer and recovered by centrifugation. Stage III, reconstitution and assay. The desorbate from Stage II is incubated with excess apoenzyme. Specific activity of the reconstituted enzyme is measured. Interpolation from a standard curve relating enzyme specific activity and pyridoxamine 5'-phosphate concentration yields the plasma level of the cofactor. Approximately 3 h are required to carry out the procedure. Much of the coenzyme was found not be assayable if plasma was refrigerated overnight or if whole blood was left standing at room temperature for a few hours. The degradation was arrested with freezing at -80 degrees C. In a 13-day experiment involving a healthy subject, sharp rises of plasma pyridoxamine 5'-phosphate were found to occur in response to small doses of oral vitamin B6.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Pyridoxamine/analogs & derivatives , Animals , Apoenzymes/isolation & purification , Apoenzymes/metabolism , Aspartate Aminotransferases/metabolism , Chromatography, Ion Exchange/methods , Eating , Fasting , Humans , Indicators and Reagents , Kinetics , Male , Middle Aged , Myocardium/enzymology , Pyridoxamine/blood , Pyridoxamine/isolation & purification , Spectrophotometry, Ultraviolet/methods , Swine
2.
Biochem Biophys Res Commun ; 151(2): 693-700, 1988 Mar 15.
Article in English | MEDLINE | ID: mdl-3348804

ABSTRACT

The cofactor activation of the apoenzyme of pig heart cytosolic aspartate aminotransferase was studied in various buffers. Cationic buffers are shown to allow maximal reconstitution in the pH range of 5.0 to 9.0. Anionic buffers made up of mono- and dicarboxylates are found to affect reconstitution in a pH-dependent manner. At low pH, the carboxylates strongly inhibit reconstitution, but at high pH, they show less effect. In contrast, the more potent inhibitor Pi shows the opposite pH profile. Dicarboxylates are considerably more inhibitory than monocarboxylates. Substantial protection against inhibition by a number of carboxylates may be achieved by the addition of sodium chloride.


Subject(s)
Aspartate Aminotransferases/metabolism , Animals , Apoenzymes/metabolism , Buffers , Cytosol/enzymology , Enzyme Activation , Hydrogen-Ion Concentration , Kinetics , Myocardium/enzymology , Swine
3.
Biochem Biophys Res Commun ; 112(2): 629-37, 1983 Apr 29.
Article in English | MEDLINE | ID: mdl-6847669

ABSTRACT

Forty compounds were surveyed for their effect on the activation of pig heart apoaspartate aminotransferase by pyridoxamine 5'-phosphate. Most of the nucleotides, sugar phosphates, coenzymes, phospholipid precursors and inorganic oxyanions tested were found to be inhibitory. With few exceptions, the only requirement for a substance to be inhibitory is the presence of a di- or polyanionic moiety analogous to the 5'-phosphate group of the cofactor. In spite of the lack of overall structural similarity to pyridoxamine 5'-phosphate, inorganic pyrophospate and apparently other inhibitors are characterized by dissociation constants comparable in magnitude to that previously reported for the natural cofactor. The physiological significance of the inhibition of coenzyme activation of apoaspartate aminotransferase by these common biological compounds is not known.


Subject(s)
Aspartate Aminotransferases/metabolism , Coenzymes/antagonists & inhibitors , Animals , Apoenzymes/metabolism , Chemical Phenomena , Chemistry , Enzyme Activation/drug effects , Kinetics , Myocardium/enzymology , Nucleotides/pharmacology , Phosphates/pharmacology , Pyridoxamine/analogs & derivatives , Pyridoxamine/pharmacology , Substrate Specificity , Sulfates/pharmacology , Swine
4.
J Med Genet ; 18(2): 156-7, 1981 Apr.
Article in English | MEDLINE | ID: mdl-7241536

ABSTRACT

A 4-month-old girl presented with myoclonic seizures and an electroencephalogram showing hypsarrhythmia. Hyperglycinuria and a cerebrospinal fluid to plasma glycine ratio of 0.2 suggested the diagnosis of non-ketotic hyperglycinaemia. Propionic acid and methyl citric acid were present in the urine, and propionyl coenzyme A carboxylase was deficient in leucocytes and fibroblasts. The ketotic and non-ketotic hyperglycinaemias cannot be differentiated by CSF: plasma glycine ratios.


Subject(s)
Carboxy-Lyases/deficiency , Glycine/blood , Myoclonus/enzymology , Female , Humans , Infant , Ketosis/enzymology , Methylmalonyl-CoA Decarboxylase , Propionates/deficiency
5.
Pediatrics ; 65(1): 107-10, 1980 Jan.
Article in English | MEDLINE | ID: mdl-7355003

ABSTRACT

A male infant who had vomiting and coma in the absence of ketoacidosis was initially thought to have dysautonomia because of abnormal responses to methacholine and histamine, as well as abnormal urinary catecholamine excretion. Following an episode of hyperammonemia, a liver biopsy was performed which revealed a partial deficiency of carbamyl phosphate synthetase activity. The patient was treated with a protein-restricted diet supplemented with a mixture of ketoacid analogues of the essential amino acids, which precipitated ketosis and acidosis. A primary deficiency of propionyl coenzyme A (CoA) carboxylase was subsequently demonstrated. Because disorders of propionate metabolism may not initially present with ketoacidosis, we recommend examination of both plasma and urine for metabolites of this pathway, as well as direct measurement of propionyl CoA carboxylase activity in peripheral blood leukocytes, before performing a liver biopsy to evaluate urea cycle enzyme activities, and particularly before adding keto acid/amino acid mixtures to a protein-restricted diet.


Subject(s)
Amino Acid Metabolism, Inborn Errors/blood , Ammonia/blood , Dysautonomia, Familial/blood , Ligases/deficiency , Acyl Coenzyme A , Amino Acid Metabolism, Inborn Errors/diet therapy , Amino Acids/therapeutic use , Carbamoyl-Phosphate Synthase (Ammonia)/blood , Carbamoyl-Phosphate Synthase (Ammonia)/deficiency , Diagnosis, Differential , Humans , Infant , Keto Acids/therapeutic use , Leukocytes/enzymology , Ligases/blood , Liver/enzymology , Male , Propionates/blood
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