Human liver high molecular weight zinc-dependent acid p-nitrophenylphosphatase. Purification and properties.
Human liver contains high molecular weight-type Zn2+-dependent acid p-nitrophenylphosphatase (HMW-ZnAP). The enzyme was purified 1000-fold by a new procedure, including preparative isoelectrofocusing. The HMW-ZnAP was homogeneous in non-denaturing disk-gel electrophoresis with an MW of about 93 kDa determined by Sephadex G-100 chromatography. A single polypeptide chain of 43 kDa was detected on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting a homodimeric structure. The isoelectric point (pI) was 7.2-7.4. Human liver HMW-ZnAP requires Zn2+-ions for activity; other divalent cations are ineffective or act as inhibitors. It dephosphorylated p-nitrophenylphosphate (pNPP) (Km = 0.24 mM), o-carboxyl phenylphosphate (oCPP) (Km = 0.92 mM) and phenylphosphate (PhP) (Km = 1.42 mM). Other substrates including [32P]-labelled casein or phosvitin, adenyl nucleotides and myo-inositol-1-phosphate, were not dephosphorylated. Human liver HMW-ZnAP obeys Michaelis-Menten kinetics with pNPP as substrate; the enzyme was competitively inhibited by inorganic phosphate (Ki = 0.55 mM), and by oCPP (Ki = 0.65 mM) and PhP (K = 1.16 mM). Adenosine monophosphate (AMP), adenosine diphosphate (ADP) and ATP displayed mixed-type inhibition. The enzyme was also inhibited by some modifiers such as EDTA, oxalate, p-chloromercurybenzoate, tartrate, imidazole, cyanide, cysteine, histidine and diethylpyrocarbonate, but not by fluoride or okadaic acid. Human liver HMW-ZnAP is sensitive to temperatures higher than 40 degrees C. The pH-dependence of the steady-state kinetic parameters indicates the existence of an essential ionizable group with a pKa of 7.25-7.50, similar to that of histidine. However, diethylpyrocarbonate inactivation experiments suggest that other amino acid residues may also be involved in enzyme catalysis.
Angiolillo
A
A
Istituto di Ricerche sulla Olivicoltura (IRO-CNR), Perugia, Italy.
Panara
F
F
D009597:4-Nitrophenylphosphatase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009597:4-Nitrophenylphosphatase; D015032:Zinc