Abstract
Abstract Pyridoxal kinase catalyzes the formation of pyridoxal phosphate, a coenzyme required for many metabolic reactions. A 2000-fold purification of the enzyme from bovine brain has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration, and affinity chromatography. Polyacrylamide gel electrophoresis at pH 9.5 and 7.5 indicates that the final product is 70 to 75% homogeneous. From sedimentation equilibrium analysis, the highly purified preparation appears to be monodisperse, and a molecular weight estimate of 80,000 has been made. A sharp pH optimum at pH 6.0 has been observed. Of the divalent metal ions studied, Zn+2 is the most effective in catalyzing the formation of pyridoxal phosphate from ATP and pyridoxal; the order of activation is Zn+2 g Co+2 g Mn+2 g Mg+2 g Fe+2. The stability constants for ZnHATP- and ZnATP-2 have been obtained at 0.3 ionic strength and 25° in the absence of Na+ or K+ by the pH titration method. The values are 1050 m-1 for ZnHATP- and 195,000 m-1 for ZnATP-2. An initial velocity study using pyridoxal as the variable substrate and ZnATP-2 as the fixed substrate gave a family of lines intersecting to the left of the vertical axis. Such an intersecting pattern is compatible with a sequential mechanism in which both pyridoxal and ZnATP-2 must add to the enzyme before pyridoxal phosphate or ADP is released.
Highlights
Of the divalent metal ions studied, B? is the most effective in catalyzing the formation of pyridoxal phosphate from ATP
In this paper we describe the purification of pyridoxal kinase from bovine brain and present initial velocity studies that suggest a sequential mechanism
The amount of pyridoxal phosphate produced was determined by reaction with phenylhydrazine according to the method of Wada and Snell [7], except that the color development reaction was allowed to proceed for only 15 min
Summary
Enzyme Assay I-Enzyme samples containing from 0.001 to 1 mg of protein were incubated at 37” for 1 hour with 1.6 mMATP, 0.16 mM Znf, and 0.32 mM pyridoxal in 0.07 M KHzPO(buffer, pH 6.5, in a total volume of 3.3 ml. Control tubes consisted of the complete assay system minus ATP. A unit of activity is defined as that amount of protein which catalyzes the formation of 1 mpmole of pyridoxal phosphate per min at 37”. At pH 6.5 and 37”, the velocity of the kinase reaction is linear for 60 min. Under these conditions, a molar ratio of 5: 0.5 : 1 (ATP-zinc-pyridoxal), which gave the maximal concentration of the ATP-zinc complex, was found to be optimal. A molar ratio of 5: 0.5 : 1 (ATP-zinc-pyridoxal), which gave the maximal concentration of the ATP-zinc complex, was found to be optimal This is in agreement with the results of Loo and Whittaker [3] for the same conditions of assay
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