PURIFICATION AND PROPERTIES OF RAT SKIN ACID PHOSPHATASES

Four acid phosphatases (APases I-IV) were purified from Triton X-100 extracts of rat skin. APase I appeared as a high-molecular weight, most likely lysosomal lipo(glyco)-protein with a pI of 4.3. APase I, extractable as a Triton-protein complex with a mol.wt. of 2 10⁶, hydrolyzed α-naphthyl phosphate (α-NP; K_m= 0.18 mM) and p-nitrophenyl phosphate (PNP; K_m= 0.96 mM) at an almost equal rate at pH 5.0. Mo₇O₂₄^6-inhibited noncompetitively (K_i= 0.15 μM). APase II had a pI of 7.8–8.0 and a mol.wt. of 104000 ± 4000. APase II is likely lysosomal and at pH 5.0 it hydrolyzed most rapidly PNP (K_m= 0.057 mM), α-NP (K_m= 0.18 mM) and phenyl phosphate (PP; K_m= 0.41 mM). Mo₇O₂₄^6- (K_i=2.5 nM) inhibited competitively. APase III, also of lysosomal origin, showed a pI of 5.3–5.5 and a mol.wt. of 87000 ± 3000. This enzyme, at pH 5.0, hydrolyzed most rapidly α-NP (K_m= 0.16 mM), pyridoxal 5-phosphate (PLP; K_m= 0.41 mM), PNP (K_m= 0.16 mM) and PP (K_m= 0.16 mM). Mo₇O₂₄^6- inhibition (K_i= 0.75 μM) was of a mixed type. p-Chloromercuribenzoate (PCMB; K_i= 2.0 μM) inhibited noncompetitively. A pI of 5.6–6.2 and a mol.wt. of 14000 ± 400 was obtained for the cytoplasmic APase IV. This enzyme, at pH 5.0, hydrolyzed most rapidly PNP (K_m= 0.16 mM), PP (K_m= 0.81 mM) and riboflavin 5′-phosphate (RP; K_m= 0.20 mM). Mo₇O₂₄^6- inhibited competitively (K_i= 8.5 μM) but PCMB (K_i= 0.32 μM) showed a mixed type of inhibition. PLP may be a natural substrate of APase III and RP that of APases II and IV. The activity of APases II and III may depend on histidyl and SH-groups, that of APase I on histidyl groups, and SH-groups may be necessary for the activity of APase IV. All these APases seem to have an active tyrosyl and carboxyl residue. The pH dependence of the catalyses, inhibition characteristics and anatomic location showed APases I-IV to be true skin acid phosphatases.