Kidney N-acetylgalactosamine (GalNAc)-1-phosphate kinase, a new pathway of GalNAc activation.
A new enzyme that phosphorylates GalNAc at position 1 to form GalNAc-alpha-1P was purified approximately 1275-fold from the cytosolic fraction of pig kidney, and the properties of the enzyme were determined. The kinase is quite specific for GalNAc as the phosphate acceptor and is inactive with GlcNAc, ManNAc, glucose, galactose, mannose, GalN, and GlcN. This enzyme is clearly separated from galactokinase by chromatography on phenyl-Sepharose. The GalNAc kinase has a pH optimum between 8.5 and 9.0 and requires a divalent cation in the order Mg2+ > Mn2+ > Co2+, with optimum Mg2+ concentration at approximately 5 mM. The enzyme was most active with ATP as the phosphate donor, but slight activity was observed with ITP, acetyl-P, and phosphoenolpyruvate. Enzyme activity was highest in porcine and human kidney and porcine liver, but was low in most other tissues. Cultured HT-29 cells also had high activity for this kinase. The purified enzyme fraction was incubated with azido-[32P]ATP, exposed to UV light, and run on SDS gels. A 50-kDa protein was labeled, and this labeling showed saturation kinetics with increasing amounts of the probe and was inhibited by unlabeled ATP. Although the most purified GalNAc kinase preparation still had two bands that labeled with ATP, maximum labeling of the 50-kDa protein, but not the 66-kDa band, was coincident with maximum GalNAc kinase activity on a column of DEAE-Cibacron blue. On Sephacryl S-300, the native enzyme has a molecular mass of 48-51 kDa, indicating that the active kinase is a monomer. The product of the reaction was characterized as GalNAc-alpha-1-P by various chemical procedures.
Pastuszak
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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Drake
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Elbein
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D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D008970:Molecular Weight; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D014018:Tissue Distribution
D002413:Cations, Divalent; D013439:Sulfhydryl Reagents; C101167:N-acetylgalactosamine-1-phosphate kinase; D017853:Phosphotransferases (Alcohol Group Acceptor); D000116:Acetylgalactosamine