Rat Liver Acid Phosphatase Research Articles

Immunocytochemical localization of acid phosphatase in rat liver.

Localization of acid phosphatase (ACPase) in rat liver was investigated by immunocytochemical techniques. Rat liver was fixed by perfusion and cut into thick tissue slices, which were embedded in Epon or Lowicryl K4M. For light microscopy (LM), semithin Epon sections were stained for the enzyme ACPase by an indirect immunoenzyme technique. For electron microscopy (EM), ultra-thin Lowicryl K4M sections were stained by a protein A-gold technique. By means of LM, granular reaction deposits were observed in hepatocytes and sinus-lining cells. Stained granules were present in the juxtanuclear cytoplasm, but they did not correspond to a typical staining pattern for the Golgi complex. EM revealed that gold particles indicating ACPase antigens were present on lysosomes and on some vesicles locating in the trans Golgi region. Endosomelike vesicles were strongly positive for the labeling. Golgi cisterna were mostly negative, but weak signals were noted in dilated sacules. The plasma membranes on the sinusoidal and bile canalicular sides were labeled by a few gold particles. The results indicate that ACPase is present in endosomes and in a restricted area of plasma membrane, as well as in the lysosomal system.

Stimulatory effect of mevinolin on rat liver phosphatidic acid phosphatase

The activity of the soluble form of phosphatidic acid phosphatase in rat liver was stimulated about 2.5-fold by inclusion of mevinolin, a competitive hydroxymethylglutaryl-CoA reductase inhibitor, in the diet (0.1%). The stimulatory effect of mevinolin was present also after dietary addition of cholestyramine (5%) or intraperitoneal administration of ethanol. Addition of cholesterol (2%) to the diet totally abolished the stimulation by mevinolin on phosphatidic acid phosphatase. The results support a correlation between the synthesis of the rate-limiting enzyme in cholesterol biosynthesis and the activity of the apparent rate-limiting enzyme in triacylglycerol biosynthesis.

Reactive thiol groups in rat liver acid phosphatase.

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.

Evidence for reactive groups in rat liver acid phosphatase: inactivation by diethyl pyrocarbonate and photo-oxidation in the presence of Rose Bengal

Diethyl pyrocarbonate and photo-oxidation have been shown to be useful agents for investigating the importance of histidine residues in proteins. The reactions involved occur under mild conditions. We report the effects of these reactions on the activity of rat liver acid phosphatase. The enzyme was prepared from rat liver by using a modification of the method of Igarashi & Hollander (1968). Two peaks of enzyme activity were obtained by chromatography on DEAE-cellulose, The peaks are designated PI and P, according to the order in which they are eluted from the column (Navaratnam et al., 1982). After further purification the specific activities of enzymes P, and P, were 57.3 and 167pmol/min per mg of protein respectively. Both enzymes PI and P, were homogeneous on polyacrylamide-gel electrophoresis. They were stable for several weeks when stored at 0 4 ° C . From a study of the variation in the rate of inactivation by diethyl pyrocarbonate with pH, it was found that the greatest rate occurred at approx. pH 8 in Hepes [4-(2-hydroxyethyl)1 -piperazine-ethanesulphonic acidlbuffer. To test the effect of diethyl pyrocarbonate on the enzymic activity, portions of enzymes PI and P, were each incubated at 20°C with the inhibitor ( C k 3 0 m ~ ) in 0.1 ~ H e p e s buffer, pH 8. The protein concentrations were 15-2Opg in the reaction mixtures. At suitable time intervals, samples were removed and assayed for residual acid phosphatase activity at 30°C and pH5 (Navaratnam et al., 1981). The protective action afforded by the substrate analogue P, was tested by including various concentrations of the anion in the inhibitor-containing mixtures. From the results shown in Fig. 1 it is evident that diethyl pyrocarbonate potently inactivates rat liver acid phosphatase, giving over 9096 inhibition after approx. 5min exposure to the highest concentration of reagent tested. The rate of inactivation was pseudo-first3rder and dependent on the concentration of the reagent. The action of the inhibitor is probably on an essential histidine residue at, or close to, the active site, since the competitive inhibitor P, at concentrations up to lOmM protected both enzymes PI and P, considerably against diethyl pyrocarbonate (results not shown). Photo-oxidation was performed at 0°C in stirred 0.1 M-Hepes buffer, pH8, containing Rose Bengal (0-13pg/ml) and 1520pg/ml of enzyme P,. A slide projector was used as the light source and positioned 20 cm from the reaction vessel. Illumination was from the side. At suitable time intervals, samples were withdrawn for assay of residual acid phosphatase activity. A pseudo-first-order loss of activity occurred that amounted to approx 90% by 4 min of illumination. The demonstration of the likely importance of histidine residues in two fractions of acid phosphatase from rat liver, together with the observations by other workers studying rat liver (Igarashi et al., 1970), bovine liver (Lawrence & van Etten, 1981), and human liver and prostate (Saini & van Etten, 1978) suggests that histidine is an essential component of the active site of all non-specific acid phosphatases. This may be an important feature that distinguishes them from alkaline phosphatases. From earlier studies it appears that rat liver has important arginine and cysteine residues in the active site (Navaratnam et ol., 1981, 1982). It will be interesting to learn the relative positions of these important residues in the native enzyme structure. -0-0-0 0

Reaction of rat liver acid phosphatase with p-chloromercuribenzoate

Reaction of rat liver acid phosphatase with p-chloromercuribenzoate

Apparent phosphorylation — dephosphorylation of soluble phosphatidic acid phosphatase in rat liver

Preincubation with alkaline phosphatase stimulated the activity of ammonium sulfate precipitated phosphatidic acid phosphatase from rat liver cytosol. Treatment with Mg-ATP inhibited the activity. The possibility is discussed that the soluble phosphatidic acid phosphatase, assumed to catalyze the rate-limiting step in the biosynthesis of triglycerides, is to some extent regulated by phosphorylation — dephosphorylation.

Evidence for reactive groups in rat liver acid phosphatase: inactivation by henylglyoxal

Evidence for reactive groups in rat liver acid phosphatase: inactivation by henylglyoxal

Effect of a transitory ischaemia on the structure-linked latency of rat liver acid phosphatase and beta-galactosidase.

The structure-linked latency of acid phosphatase and beta-galactosidase was studied in rat liver lobes made ischaemic for 1 or 2 h and then recirculated with blood for increasing periods. Free activity of acid phosphatase and unsedimentable activity of beta-galactosidase are increased in homogenates of ischaemic livers. When ischaemia had been maintained for 1 h, the recovery of normal latency for both enzymes was observed 1 h after re-establishment of the blood flow. After a 2 h period of ischaemia, unmasked activity markedly decreases during the first 1 h after restoration of blood flow; after that, a large and irreversible secondary rise takes place. Chlorpromazine, injected 30 min before or just after induction of ischaemia, extensively prevents the latency decrease occurring during restoration of blood flow. Modifications of the hydrolase distribution pattern obtained after differential centrifugation are in agreement with the latency changes. These results suggest that a 2 h ischaemia causes an alteration of the liver lysosomes that is largely reversible and that restoration of blood flow induces an irreversible alteration of these organelles. Chlorpromazine treatment prevents the irreversible lesion from taking place.

Influence of bile acids on the soluble phosphatidic acid phosphatase in rat liver

Summary Biliary drainage was found to increase soluble phosphatidic acid phosphatase activity in rat liver about two-fold. Treatment with cholestyramine increased the same activity with about 70%. Treatment with 1% cholic acid or chenodeoxycholic acid decreased the activity 76% and 93%, respectively. It is suggested that the enterohepatic circulation of bile acids might be of some regulatory importance for biosynthesis of triglycerides in the liver. The findings are discussed in relation to previous clinical observations that changes in bile acid metabolism affect plasma triglyceride turnover.

Histochemical acitivity of acid phosphatase in rat liver after perfusion fixation.

Acid phosphatase activity was studied histochemically in rat livers after perfusion fixation. The three different perfusates used were (1) 2% of paraformaldehyde; (2) 1.25% of glutaraldehyde, and (3) 1.25% of each of paraformaldehyde and glutaraldehyde. Unfixed fresh frozen sections showed high enzymatic activity in both centrilobular (CL) and periportal (PP) areas. A similar localization pattern resulted after perfusate 1 indicating a lack of reduction in the enzymatic activity in either of the two areas. After perfusate 2, PP arease remained intensely stained while slight reduction of the enzymatic activity was exhibited by the CL regions. Perfusion (3) resulted in higher activity in PP areas but maximal inhibition in the CL region. The inhibition of acid phosphatase activity in the CL areas correlates with regional differences of Kupffer cell population as well as the existence of metabolic heterogeneity between the two hepatic regions.

Dimeric nature and amino acid compositions of homogeneous canine prostatic, human liver and rat liver acid phosphatase isoenzymes. Specificity and pH-dependence of the canine enzyme

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p- nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000, respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found for mol of isoenzyme 1 and 18 for isoenzyme II.

Effect of dieldrin on the stability of lysosomes in the rat liver.

The effect of dieldrin on the bound and free activity of cathepsin, acid ribonuclease, acid phosphatase and aryl sulphatase has been investigated. Administration of dieldrin increased the activity of free cathepsin, acid ribonuclease, and acid phosphatase in rat liver whereas it did not affect the activity of aryl sulphatase. This indicates that dieldrin labilizes the lysosomes.

Comparative determination of liver acid phosphatase activity in decapitated and perfused rats.

Rat liver acid phosphatase activity in perfused and decapitated animals was analysed. The perfusion technique significantly increased the reproducibility of the results, whereas with the decapitation method a nonsystematic fault was introduced. Furthermore the results in the decapitated group have always to be interpreted as the sum of blood and tissue enzyme activity.

Acid Phosphatase from Rat Liver: Purification, crystallization, and properties

Rat liver acid phosphatase (EC 3.1.3.2) was separated into two highly purified fractions, differing in isoelectric point and Km. One fraction was crystallized and proved homogeneous by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weights (100,000), substrate specificities, and pH optima of both enzymes were similar. Oxalate was a mixed type inhibitor to both enzymes. In the presence of dioxane, both enzymes exhibited sigmoidal inhibition curves by oxalate and by trypan blue.

Rat liver acid phosphatase: differences in lysosomal and cytoplasmic forms.

SummaryTwo acid phosphatases, a lyso-somal and supernatant form, have been demonstrated in rat liver by differences in their heat stabilities at 50dGC, pH activity curves and sensitivities to tartrate and fluoride inhibition.