Modulatory effect of divalent metal cations on the phosphotyrosine activity of the frog liver acid phosphatase.

Frog liver acid phosphatase hydrolyzes phosphotyrosine at acidic pH optimum. Mn2+, Ca2+ and Mg2+ (but not Zn2+) ions modulate this activity by shifting its pH optimum to physiological pH. This effect is not observed when p-nitrophenylphosphate is used as a substrate. Phosphoserine and phosphothreonine are not hydrolyzed under the same conditions.

A novel 35 kDa frog liver acid metallophosphatase.

The lower molecular weight (35 kDa) acid phosphatase from the frog (Rana esculenta) liver is a glycometalloenzyme susceptible to activation by reducing agents and displaying tartrate and fluoride resistance. Metal chelators (EDTA, 1,10-phenanthroline) inactivate the enzyme reversibly in a time- and temperature-dependent manner. The apoenzyme is reactivated by divalent transition metal cations, i. e. cobalt, zinc, ferrous, manganese, cadmium and nickel to 130%, 75%, 63%, 62%, 55% and 34% of the original activity, respectively. Magnesium, calcium, cupric and ferric ions were shown to be ineffective in this process. Metal analysis by the emission spectrometry method (inductively coupled plasma-atomic emission spectrometry) revealed the presence of zinc, iron and magnesium. The time course of the apoenzyme reactivation, the stabilization effect and the relatively high resistance to oxidizing conditions indicate that the zinc ion is crucial for the enzyme activity. The presence of iron was additionally confirmed by the visible absorption spectrum of the enzyme with a shoulder at 417 nm and by the electron paramagnetic resonance line of high spin iron(III) with geff of 2.4. The active center containing only zinc or both zinc and iron ions is proposed. The frog liver lower molecular weight acid phosphatase is a novel metallophosphatase of lower vertebrate origin, distinct from the mammalian tartrate-resistant, purple acid phosphatases.

The 102 kDa chicken liver acid phosphatase: dimeric structure, glycoprotein nature and immunological properties.

In this study we isolated a highly purified, homogeneous high molecular weight (HMW) AcPase (Mr 102 kDa) from the chicken liver. This enzyme was shown to be a slightly acidic (pI 5.0-6.1), dimeric sialoglycoenzyme, composed of two equivalent subunits. Its sugar moiety, characterized by interactions with specific lectins, was shown to be composed of hybrid and complex type of carbohydrate chains. Heterogeneity of the high molecular weight AcPase arising from variations of the sugar components was demonstrated by isoelectric focusing, followed by reactions of the isoelectric components with specific lectins on NC membranes. Structural relationship based on immunological similarities was shown between the HMW AcPases from carp, frog, and chicken livers.

Characterization of the type of carbohydrate chains of the higher molecular weight (140 kDa) acid phosphatase of the frog liver.

1. The higher molecular weight, (HMW, M(r) 140 kDa) acid phosphatase (AcPase) of the frog liver (Rana esculenta) was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient and their carbohydrate chains were analyzed by specific lectin binding after native blotting. 2. The lectin-binding patterns obtained with ConA, WGA, LcH and PNA as well as with WGA and PNA after desialylation indicate that the frog liver HMW AcPase contains predominantly N-linked complex and/or hybride type carbohydrate chains with terminal sialic acid and fucose residues; O-glycosylated enzyme components with free and sialic acid substituted Gal-GalNAc sequences were also detected.

Amino acid composition and immunochemical properties of AcPase III and AcPase IV representing glycoforms of the lower molecular weight, tartrate-resistant acid phosphatase of the frog liver.

1. Amino acid composition and immunological properties of the frog liver LMW AcPase forms: AcPase III and IV were examined. 2. AcPase III and IV show nearly identical amino acid composition and close immunological similarity. 3. These results indicate protein identity of both the enzyme forms and together with our previous data [Kubicz A., Szalewicz A. and Chrambach A., Int. J. Biochem. 23, 413-419 (1991)] demonstrate that generation of AcPase III and IV is a modification of the same enzyme protein by glycosylation processes. 4. Differences in immunoreactivity between AcPase III and IV were observed and discussed to be due to their altered conformations.

Studies on the oligosaccharide heterogeneity of the isoelectric forms of the lower molecular weight acid phosphatase of frog liver.

1. The lower molecular weight, heterogeneous acid phosphatase (AcPase) from the frog liver (Rana esculenta) containing AcPase I, II, III and IV was separated into enzymatically active components by isoelectric focusing in an immobilized pH gradient. 2. The blotted enzyme bands were characterized by their different binding patterns obtained with the lectins concanavalin A, wheat germ agglutinin (WGA), Lens culinaris hemagglutinin (LcH) and peanut agglutinin (PNA). 3. In situ neuraminidase treatment reduced the staining intensity of some WGA-bands and increased that of PNA-bands. 4. The finding that AcPases I, II, III and IV differ in their carbohydrate chain composition, together with previous results showing different bioactivities of AcPases III and IV, indicates a correlation between the glycosylation state of enzyme forms and their physiological action.

Electrofocusing of acid phosphatases from frog liver, using an immobilized pH gradient.

Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140,000) and the lower molecular weight (LMW, Mr 38,000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; b) preventing evaporation from the gel and c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Atland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.

The lower molecular weight acid phosphatase from the frog liver: isolation of homogeneous AcPase III and IV representing glycoforms with different bioactivity.

1. AcPase III and AcPase IV, the major enzyme forms of the LMW AcPase of the frog (Rana esculenta) liver were resolved and purified to homogeneity. 2. AcPase III and IV showed a single protein band on SDS-PAGE corresponding to a mol. wt (Mr) of about 35,000. The Mr of the native enzyme forms were 33,200 (gel electrophoresis) and 38,200 +/- 5000 (gel filtration). This indicates that they are monomeric proteins sharing the same protein molecule. 3. AcPase III and IV differ essentially in thermostability and the activating effect of ConA binding. 4. AcPase III and IV are considerably activated with DTT but they differed markedly by the extent of this activation and the accompanying changes of their pH-activity curves. 5. It is suggested that the frog liver LMW AcPase represents a set of glycoforms whose different bioactivity is determined by the redox states of their essential cysteine residues.

The high molecular weight and the low molecular weight acid phosphatases of the frog liver and their phosphotyrosine activity.

1. Two molecular weight classes of non-specific acid phosphatases (AcPases) (3.1.3.2) are present in the frog (Rana esculenta) liver: a higher molecular weight (HMW) of Mr 140,560 and a lower molecular weight (LMW) of Mr 38,180 enzyme. 2. The LMW AcPase was described earlier and the HMW AcPase of optimum pH 4.8 is shown to be a L(+)-tartrate sensitive, thermolabile, dimeric glycoenzyme slightly activated by DTT. 3. The HMW and the LMW AcPases exhibit activity for phosphotyrosine which showed similar sensitivity to various effectors as the p-nitrophenyl phosphatase activity; however, both enzymes differed substantially in this respect suggesting that they might be involved in different metabolic steps.