Comparison of the antigenic peptides between human prostatic and lysosomal acid phosphatases

The relative antigenicity (capacity to bind antibodies raised against the intact prostatic acid phosphatase) of the selected peptides from human prostatic and lysosomal acid phosphatases was evaluated in a competitive assay. Both prostatic and lysosomal acid phosphatases were shown to possess similar antigenic determinants on both terminal regions, along with more similarity on NH₂-terminal peptide than COOH-terminal site. At least one additional antigenic site is present at the internal region of prostatic acid phosphatase, since the mixture of both amino- and carboxyl-terminal peptides exhibited only 70% inhibition.     

A prostatic-like acid phosphatase is present in human lactating milk

Summary The presence of a prostatic-like acid phosphatase is reported in human lactating milk. Its activity is associated with skim milk and it could be separated from the other acid phosphatases only after Triton X-100 treatment. By all the criteria applied, it appears to be very similar to prostatic acid phosphatase. An approximate molecular weight of 96 000 was measured for the native enzyme, which is inhibited by L-(+)tartrate and has similar electrophoretic migration. Besides, it hydrolyzes choline-o-phosphate very well and cross-reacts with an antibody anti-prostatic acid phosphatase. This prostatic-like acid phosphatase has also been detected in a human mammary carcinoma from a lactating patient.     

Prostatic-like acid phosphatase in human endometrial glands and its cyclic activity

Estrogen-induced autocrine and paracrine growth factors are thought to stimulate endometrial proliferation. However, the proliferation is arrested at an early secretory phase although the amount of growth factors and their receptors remains constant. These receptors are protein tyrosine kinases which cause activating receptor autophosphorylation and phosphorylation of signalling substances. One inhibitory mechanism is the reverse dephosphorylation by phosphatases hydrolysing phosphotyrosines. Previously, an acid phosphotyrosine phosphatase activity was found in endometrial secretory glands. The purpose of this study was to evaluate its characteristics. Catalytic and immunohistochemical techniques were applied on sections obtained from human endometrium and other tissues. Endometrial acid phosphatase hydrolysed phosphotyrosine, not only at acid, but also at neutral pH values. An alternative substrate was α-naphthyl phosphate or β-glycerophosphate but not phosphoserine. Activities were inhibited by tartrate and fluoride but not by formaldehyde. These catalytic properties are identical only to those of prostatic acid phosphatase (PAP). A PAP-like nature was also proved by positive PAP immunohistochemistry. In conclusion, endometrial glands contain a phosphotyrosine phosphatase which is identical to PAP. Its activity is menstrual-cycle-dependent, being present only at the secretory phase, and it may counterbalance receptor tyrosine kinases terminating glandular proliferation despite constant levels of growth factors and their receptors.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5-phosphate (K=21M) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5-phosphate inhibition has been proposed.Abbreviations used PTPase phosphotyrosine protein phosphatase - TFA trifluoroacetic acid - SDS sodium dodecylsulfate - T tryptic peptides - SP endoproteinase Glu-C peptides - FAB fast atom bombardment - Ac acetyl - HPLC high-performance liquid chromatography - OPA o-phtaldialdehyde - PMSF phenylmethylsulfonyl fluoride - CD45 leukocyte common-antigen PTPase - LAR leukocyte-antigen-related PTPase - PTP IB human placental PTPase     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

Studies of the purine analog associated modulation of human erythrocyte acid phosphatase activity

Summary The activity of the human erythrocyte acid phosphatase is modulated by a series of structural analogs of purine. The unsubstituted purine base does not affect the enzyme activity. Addition of a substituent at the number six position usually generates an analog which activates the enzyme while similar substitutions at the two position usually generate an inhibitor. Pyrimidines are generally ineffective as modulators while several modifications of the imidazole ring of the purine analogs do not abolish the modulator activity of the purine analog. The level of response to all active analogs is isozyme specific. Differences in apparent relative affinities among the modulators are noted. The modulators with a positive effect on enzyme activity, are effective in the presence of methanol which is more effective than H₂0 as a phosphate acceptor. These analogs act by enhancing the rate of transfer of phosphate to H₂O, while decreasing the rate of transfer to methanol. The results suggest that the purine analogs may act by altering the rate of hydrolysis of the phosphoenzyme intermediate by H₂0 or may change the rate-limiting step in the catalytic mechanism.     

Direct observation of multiple protonation states in recombinant human purple acid phosphatase

To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK _a,1), an active form (pK _a,1<pH<pK _a,2), and an inactive high pH form (pH>pK _a,2). The pK _a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.Electronic Supplementary Material Supplementary material is available for this article at     

Plasmid DNA vaccine encoding prostatic acid phosphatase is effective in eliciting autologous antigen-specific CD8+ T cells

Prostatic acid phosphatase (PAP) is a prostate cancer tumor antigen and a prostate-specific protein shared by rats and humans. Previous studies indicated that Copenhagen rats immunized with a recombinant vaccinia virus expressing human PAP (hPAP) developed PAP-specific cytotoxic T cells (CTL) with cross reactivity to rat PAP (rPAP) and evidence of prostate inflammation. Viral delivery of vaccine antigens is an active area of clinical investigation. However, a potential difficulty with viral-based immunizations is that immune responses elicited to the viral vector might limit the possibility of multiple immunizations. In this paper, we investigate the ability of another genetic immunization method, a DNA vaccine encoding PAP, to elicit antigen-specific CD8+ T cell immune responses. Specifically, Lewis rats were immunized with either a plasmid DNA-based (pTVG-HP) or vaccinia-based (VV-HP) vaccine each encoding hPAP. We determined that rats immunized with a DNA vaccine encoding hPAP developed a Th1-biased immune response as indicated by proliferating PAP-specific CD4+ and CD8+ cells and IFNγ production. Rats immunized with vaccinia virus encoding PAP did not develop a PAP-specific response unless boosted with a heterologous vaccination scheme. Most importantly, multiple immunizations with a DNA vaccine encoding the rat PAP homologue (pTVG-RP) could overcome peripheral self-tolerance against rPAP and generate a Th1-biased antigen-specific CD4+ and CD8+ T cell response. Overall, DNA vaccines provide a safe and effective method of generating prostate antigen-specific T cell responses. These findings support the investigation of PAP-specific DNA vaccines in human clinical trials.     

Heparin column analysis of serum type 5 tartrate-resistant acid phosphatase isoforms

The objective of the present study was to develop a specific method for the separation of tartrate-resistant acid phosphatase (TRAP) derived exclusively from osteoclasts. Heparin column-bound TRAP in human serum was separated into three peaks of TRAP activity when eluted with a linear gradient of sodium chloride. The last peak corresponded to TRAP 5b which was first named according to its electrophoretic mobility [Clin. Chem. 24 (1978) 309] and was considered to be derived from osteoclasts [J. Bone Miner. Res. 13 (1998) 683]. The second peak was found to be TRAP 5a. The height of the last peak varied from age to age.     

Cellular prostatic acid phosphatase,a PTEN-functional homologue in prostate epithelia,functions as a prostate-specific tumor suppressor

The inactivation of tumor suppressor genes (TSGs) plays a vital role in the progression of human cancers. Nevertheless, those ubiquitous TSGs have been shown with limited roles in various stages of diverse carcinogenesis. Investigation on identifying unique TSG, especially for early stage of carcinogenesis, is imperative. As such, the search for organ-specific TSGs has emerged as a major strategy in cancer research. Prostate cancer (PCa) has the highest incidence in solid tumors in US males. Cellular prostatic acid phosphatase (cPAcP) is a prostate-specific differentiation antigen. Despite intensive studies over the past several decades on PAcP as a PCa biomarker, the role of cPAcP as a PCa-specific tumor suppressor has only recently been emerged and validated. The mechanism underlying the pivotal role of cPAcP as a prostate-specific TSG is, in part, due to its function as a protein tyrosine phosphatase (PTP) as well as a phosphoinositide phosphatase (PIP), an apparent functional homologue to phosphatase and tensin homolog (PTEN) in PCa cells. This review is focused on discussing the function of this authentic prostate-specific tumor suppressor and the mechanism behind the loss of cPAcP expression leading to prostate carcinogenesis. We review other phosphatases' roles as TSGs which regulate oncogenic PI3K signaling in PCa and discuss the functional similarity between cPAcP and PTEN in prostate carcinogenesis.     

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes

A study has been made of the decay of acid phosphatase (ACP₁) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.     

Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.     

Molecular cloning of cDNA for human prostatic acid phosphatase

A human liver cDNA library in λgt11 was screened with polyclonal antiserum to human acid phosphatase isoenzyme 2a/4. About eleven positive clones have been obtained. Two clones, λ Hap21 and λ Hap22 were further characterized: clone λHap21 contained a 0.8-kb cDNA insert and clone λHap22 a 1.8–2.0-kb insert. XbaI digestion of λHap22 generated two fragments of 1.0 and 0.9 kb. BglII digestion resulted in a 1.2-kb fragment and several smaller fragments of undetermined size. Clone 1 Hap22 contained all the genes carried by λ gt11(lac 5cI857nin 5Sam 100) and the 2-kb insert. An Escherichia coli(λHap22) lysogen was generated, and its acid phosphatase activity was approximately ten-fold higher than that in the control nonlysogenic lysate. Western-blot analysis of total proteins present in this E. coli(λHap22) lysate revealed that the non-induced λHap22 prophage directed the synthesis of an approx. 175-kDa protein. This protein was recognized by antibody to the human acid phosphatase isoenzyme 2a/4 and anti-β-galactosidase and was produced only upon induction with IPTG. These results indicated that AHap22 carried a major portion of the gene coding for the human acid phosphatase isoenzyme 2a and/or 4 and this protein fragment of acid phosphatase was sufficient to manifest enzymatic activity.     

Polymorphism of acid phosphatase in chicken leucocytes

Three individual variants of acid phosphatase in chicken leucocytes were found by means of starch gel electrophoresis. The phenotype in leucocytes showed the same appearance as polymorphic forms of liver acid phosphatase in the same bird. The study of the Hardy-Weinberg distribution of the phenotypes of acid phosphatase in leucocytes also indicated that they are controlled by the same pair of codominant autosomal alleles as the phenotypes in the liver.
Acid phosphatase is polymorphic in all six strains of chickens studied.     

Effect of vanadium compounds on acid phosphatase activity

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.     

Identification of caffeoylquinic acid derivatives as natural protein tyrosine phosphatase 1B inhibitors from Artemisia princeps

Considerable attention has been paid to protein tyrosine phosphatase 1B (PTP1B) inhibitors as a potential therapy for diabetes, obesity, and cancer. Ten caffeoylquinic acid derivatives (1–10) from leaves of Artemisia princeps Pamp. (Asteraceae) were identified as natural PTP1B inhibitors. Among them, chlorogenic acid (3) showed the most potent inhibitory activity (IC₅₀ 11.1?μM). Compound 3 was demonstrated to be a noncompetitive inhibitor by a kinetic analysis. Molecular docking simulation suggested that compound 3 bound to the allosteric site of PTP1B. Furthermore, compound 3 showed remarkable selectivity against four homologous PTPs. According to these findings, compound 3 might be potentially valuable for further drug development.     

The discovery of the fat-regulating phosphatidic acid phosphatase gene

Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid (substrate) and diacylglycerol (product), which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol. Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression, membrane expansion, secretion, and endocytosis. While this important enzyme has been known for several decades, its gene was only identified recently from yeast. This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans.     

Release of nucleotide-cleaving acid phosphatase from carrot cells grown in suspension culture

Carrot ( Daucus carota L. cv. Lunga di Amsterdam) cells grown in suspension culture release into the culture medium a phosphatase capable of converting deoxyribo- as well as ribonucleoside triphosphates into nucleosides. The enzyme activity requires acidic pH and allows a prompt utilization of phosphorylated DNA precursors as measured by tritiated dTTP incorporation. Such utilization is partially inhibited by inorganic phosphate and completely inhibited by ATP.