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 per mol of isoenzyme I and 18 for isoenzyme II.     

Human prostatic acid phosphatase: cDNA cloning, gene mapping and protein sequence homology with lysosomal acid phosphatase

The cDNAs encoding human prostatic acid phosphatase were cloned and characterized. The mRNAs contain 3' noncoding regions of heterogeneous sizes 646, 1887 or 1913 nucleotides. A dimer and a monomer of the conserved Alu-repeats are present in the longer 3' noncoding sequences. The complete sequence of 354 amino acids for the mature enzyme was determined by sequencing both cDNA and protein. Human prostatic and lysosomal acid phosphatases exhibit 50% sequence homology, including five Cys residues and two putative N-linked glycosylation sites. The Acp-3 gene coding for human prostatic acid phosphatase was mapped onto chromosome 3 in this investigation. The Acp-2 gene coding for lysosomal acid phosphatase has previously been located on chromosome 11, while the Acp-1 gene coding for red blood cell acid phosphatase is on chromosome 2.     

Stereoselectivity of binding of alpha-(N-benzylamino)benzylphosphonic acids to prostatic acid phosphatase

The inhibition effects of enantiomerically pure alpha-(N-benzylamino)benzylphosphonic acids and their derivatives on human prostatic acid phosphatase have been investigated. As expected, (R)-alpha-(N-benzylamino)benzylphosphonic acid demonstrated higher affinity for the enzyme than (S)-enantiomer. At the same time, (1R,2S)-phenyl[(1-phenylethyl)amino]methylphosphonic acid was found to be a significantly weaker inhibitor than its (1S,2R)-analogue. The enantioselectivity has been explained using a molecular modeling approach by computational docking of inhibitors into active center of prostatic acid phosphatase.     

The Deceptive Acid Phosphatases

Determination of the prostatic acid phosphatase is, theoretically, a specific test for carcinoma of the prostate, but the present laboratory techniques have produced too many false positives and false negatives to be dependable. There may be inhibitors or enzymes that interfere with these tests.Until more exact enzymes are discovered, the present acid phosphatases should not be depended upon as a criterion for the type of surgical operation in carcinoma of the prostate, nor, without biopsy, should they be taken as an indication of prostatic malignant disease.     

Comparative investigation of the mixed aggregation immunocytochemical technique and the indirect peroxidase technique for the detection of prostate specific acid phosphatase in paraffin or paraplast sections

Summary The results obtained with the indirect peroxidase technique for the identification of prostate specific acid phosphatase in formalin fixed, paraffin or paraplast embedded autopsy material are compared with the results obtained with the mixed aggregation immuno-cytochemical technique. When using a monospecific antiserum the former technique is prefered. However, when a monospecific antiserum is not available, one has to balance the advantages of the mixed aggregation immuno-cytochemical technique against the disadvantages of having to prepare a monospecific antiserum, necessary for the indirect peroxidase technique. Both methods appeared positive in 20 prostatic carcinomas and in 36 metastases of prostatic carcinomas. In the epithelium of the seminal vesicles and in osteoclasts no acid phosphatase could be detected with the antiserum. A comparison of both techniques, as well as different types of preincubation to diminish nonspecific background staining are discussed.     

Comparative investigation of the mixed aggregation immunocytochemical technique and the indirect peroxidase technique for the detection of prostate specific acid phosphatase in paraffin or paraplast sections.

The results obtained with the indirect peroxidase technique for the identification of prostate specific acid phosphatase in formalin fixed, paraffin or paraplast embedded autopsy material are compared with the results obtained with the mixed aggregation immuno-cytochemical technique. When using a monospecific antiserum the former technique is prefered. However, when a monospecific antiserum is not available, one has to balance the advantages of the mixed aggregation immuno-cytochemical technique against the disadvantages of having to prepare a monospecific antiserum, necessary for the indirect peroxidase technique. Both methods appeared positive in 20 prostatic carcinomas and in 36 metastases of prostatic carcinomas. In the epithelium of the seminal vesicles and in osteoclasts no acid phosphatase could be detected with the antiserum. A comparison of both techniques, as well as different types of preincubation to diminish nonspecific background staining are discussed.     

Prostatic Contribution to Normal Serum Acid Phosphatase

Total and tartrate-labile serum acid phosphatase levels were compared in patients with and without prostates, and in 12 patients before and after cystoprostatectomy. Absence of the prostate seems to make no significant difference to the levels of serum acid phosphatase. There is no justification for referring to the tartrate-labile serum acid phosphatase as “prostatic acid phosphatase.” A substantial incidence of marginally raised levels of serum acid phosphatase in each group of patients suggests that the upper limit of normal for the total serum acid phosphatase should be taken as 5 K.A.u.     

Proteomic analysis of formalin-fixed prostate cancer tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.     

Increase in acid phosphatase activity in the fat body during larval and pharate pupal development in Calliphora erythrocephala

Biochemical evidence was obtained for an increase in acid phosphatase activity in the larval fat body of Calliphora erythrocephala during larval and pharate pupal instars. This observation is in conflict with published data indicating a decreasing enzyme activity in late third stage larvae. Centrifugation and filtration studies showed that the pH of the homogenisation medium has a strong influence on the solubilisation of acid phosphatase and its distribution in homogenate components. Differences in biochemical techniques including the pH value may explain the discrepancy between the published results and the present findings.The observed increase in acid phosphatase activity is related to the activity of the lysosomal system in the period immediately preceding pupal-adult apolysis.     

Prostatic acid phosphatase, a neglected ectonucleotidase

Two recent papers reveal that the soluble and secreted prostatic acid phosphatase, an enzyme that has long served as a diagnostic marker for prostate cancer, has a membrane-bound splice variant. This enzyme exhibits ecto-5′-nucleotidase activity, is widely distributed, and implicated in the formation of chronic pain. While prostatic acid phosphatase hydrolyzes phosphomonoesters other than 5′-nucleoside monophosphates these novel data suggest that, in addition to ecto-5′-nucleotidase and the alkaline phosphatases, prostatic acid phosphatase must be taken into account in future studies on extracellular adenosine production.     

The epidermal growth factor receptor from prostate cells is dephosphorylated by a prostate-specific phosphotyrosyl phosphatase.

Human prostatic acid phosphatase (PAcP) has been found to have phosphotyrosyl-protein phosphatase activity (H. C. Li, J. Chernoff, L. B. Chen, and A. Kirschonbaun, Eur. J. Biochem. 138:45-51, 1984; M.-F. Lin and G. M. Clinton, Biochem. J. 235:351-357, 1986) and has been suggested to negatively regulate phosphotyrosine levels, at least in part, by inhibition of tyrosine protein kinase activity (M.-F. Lin and G. M. Clinton, Adv. Protein Phosphatases 4:199-228, 1987; M.-F. Lin, C. L. Lee, and G. M. Clinton, Mol. Cell. Biol. 6:4753-4757, 1986). We investigated the molecular interaction of PAcP with a specific tyrosine kinase, the epidermal growth factor (EGF) receptor, from prostate carcinoma cells. Of several proteins phosphorylated in membrane vesicles from prostate carcinoma cells, PAcP selectively dephosphorylated the EGF receptor. The prostate EGF receptor was more efficiently dephosphorylated by PAcP than by another phosphotyrosyl phosphatase, potato acid phosphatase. Further characterization of the interaction of PAcP with the EGF receptor revealed that the optimal rate of dephosphorylation occurred at neutral rather than at acid pH. Thus, the enzyme that we formerly referred to as PAcP we now call prostatic phosphotyrosyl-protein phosphatase. Hydrolysis of phosphate from tyrosine residues in the immunoprecipitated EGF receptor catalyzed by purified prostatic phosphotyrosyl-protein phosphatase caused a 40 to 50% decrease in the receptor tyrosine kinase activity with angiotensin as the substrate. In contrast, autophosphorylation of the receptor was associated with an increase in tyrosine kinase activity.     

Serum and seminal markers in the diagnosis of disorders of the genital tract of the dog: a mini-review

Serum and seminal biologic substances that are produced either by normal or abnormal tissues of the organism and that can be used to diagnose pathological conditions are usually referred as markers. The aim of this article is to briefly review the most relevant clinical features of the main genital markers in the male dog: alkaline phosphatase (AP), carnitine and canine prostate-specific arginine esterase (CPSE). Carnitine and AP are markers for the presence of epididymal fluid in the ejaculate and their measurement in azoospermic dogs has been used as an indicator of tubular patency of the ductal network. Although AP is not present in high concentrations in the testis, this does not preclude the possibility that testicular cells might secrete some AP. If this were true, AP could also reflect, at least in some degree, germ cell function in this species. Prostate-specific arginine esterase, the major secretory product of the canine prostate, is a known marker of gland secretion in the dog. Tumor markers frequently used in human medicine, such as prostatic acid phosphatase and prostate-specific antigen, are is still controversial in the diagnosis of prostatic carcinoma of the dog. Although further research is necessary to define the exact role of CPSE, it seems to be a promising diagnostic tool in nonneoplasic canine prostatic disorders. Future studies should also address the quantitative relationship among serum and prostatic androgen levels, prostatic androgen-dependent problems and how these are affected by anti-androgen treatment. The aim of this article is to briefly review the most relevant clinical features of three main genital markers of the male dog.     

Role of lymphography in carcinoma of the prostate.

The results of bilateral pedal lymphography in 83 patients with adenocarcinoma of the prostate gland are presented. The patients were divided into two groups: 45 new cases and 38 late or old cases presenting several years after the onset of the disease. Altogether 25 of the new patients and 29 of the late patients had lymphographic evidence of lymph node metastases. The lymphogram results in relation to local tumour size, histological grade, the presence of skeletal metastases, and acid phosphatase levels are discussed. Of the new patients with T1 and T2 tumors--that is, those still localized within the prostatic capsule--41% had positive lymphograms. The inaccuracy of acid phosphatase estimations in detecting early extraprostatic spread is shown and compared with the greater accuracy of lymphography. Lymphography should be used as an initial investigation in all cases where aggressive therapy is being considered, and the importance of regular follow-up radiographs is emphasized.     

Cooperative kinetics of human prostatic acid phosphatase

The steady-state kinetics of hydrolysis reaction catalysed by human prostatic acid phosphatase (PAP) by using 1-naphthyl phosphate, phenyl phosphate and phosphotyrosine as substrates has been studied at pH 5.5. The substrate binding curves were sigmoidal and Hill cooperation coefficient h was higher than 1 for each of the examined compounds. Thus, human prostatic acid phosphatase kinetics exhibits positive cooperativity towards the studied substrates. The extent of cooperativity was found to depend on the substrate used and on enzyme concentration. The highest cooperativity of PAP was observed for 1-naphthyl phosphate and the lowest for phosphotyrosine. When prostatic phosphatase concentration increased, Hill cooperation coefficient (h) and half saturation constant (K(0.5)) both grew, but the catalytic constant (k(cat)) remained constant, for each of the substrates studied. Ligand-induced association-dissociation equilibrium of the active oligomeric species (monomer-dimer-tetramer-oligomers) is suggested.     

Probing molecular interaction between concanavalin A and mannose ligands by means of SFM

Recently, the scanning force microscope (SFM) has been widely used for direct monitoring of specific interactions between biologically active molecules. Such studies have employed the SFM liquid-cell setup, which allows measurements to be made in the native environment with force resolution down to a tenth of a picoNewton. In this study, the ligand–receptor strength of monoclonal anti-human prostatic acid phosphatase and prostatic acid phosphatase, representing an antigen–antibody system with a single type of interaction, was determined. Then, the interaction force occurring between concanavalin A and the carbohydrate component of the glycoproteins arylsulfatase A and carboxypeptidase Y was measured. High mannose-type glycans were sought on the human prostate carcinoma cell surface. Application of an analysis based on the Poisson distribution of the number of bonds formed in all these measured systems allowed the strength of the molecular interaction to be calculated. The values of the force acting between two single molecules were 530±25, 790±32, and 940±39 pN between prostatic acid phosphatase and monoclonal anti-human prostatic acid phosphatase, between concanavalin A and arylsulfatase A, and between concanavalin A and carboxypeptidase Y, respectively. The value calculated from data collected for the force between concanavalin A and mannose-containing ligands present on the surface of human prostate carcinoma cells was smaller, 116±17 pN. The different values of the binding force between concanavalin A and mannose-containing ligands were attributed to the structural changes of the carbohydrate components.     

Native blot and immunotransfer of human prostatic acid phosphatase isozymes

Agarose gel isoelectrofocusing is used to separate the isozymes of human prostatic acid phosphatase with retention of enzyme activity. The native blotting of the isozymes onto a nitrocellulose membrane increases the sensitivity of the enzyme stain and is suitable for analysis of isozymes in prostate tissue, which contains little nonprostatic acid phosphatase. The specificity of the transfer is increased by treating the membrane with antibody to human prostatic acid phosphatase prior to the transfer. The specificity of the antibody is conferred to the membrane resulting in a transfer specific for prostatic acid phosphatase. The immunotransfer procedure is applicable to serum which contains appreciable amounts of nonprostatic acid phosphatase.     

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.     

A phosphotyrosyl-protein phosphatase activity associated with acid phosphatase from human prostate gland

Using [32P]P-Tyr-IgG and [32P]P-Tyr-casein phosphorylated by pp60v-src as substrates, studies on the phosphotyrosyl-protein phosphatase activity in human prostate gland indicate that it is associated with prostatic acid phosphatase. Evidence to support this conclusion include the following: (a) these two enzymatic activities co-purify to apparent homogeneity; (b) they co-migrated on polyacrylamide gel electrophoresis, ion-exchange and gel filtration chromatographies; (c) the exhibit identical thermostability; and (d) the phosphotyrosyl-protein phosphatase activity is sensitive to inhibition by p-nitrophenyl phosphate and by several classical inhibitors of prostatic acid phosphatase including L(+)-tartrate, molybdate, vanadate and NaF. The purified enzyme exhibits high specificity towards phosphotyrosyl-proteins with little activity towards several phosphoseryl-proteins and phosphothreonyl-proteins examined. The present findings indicate that prostatic acid phosphatase may function in vivo as a phosphotyrosyl-protein phosphatase.     

Steroid hormone regulation of prostatic acid phosphatase expression in cultured human prostatic carcinoma cells

We have investigated the modulation of prostatic acid phosphatase expression in the human prostatic cancer cell line LNCaP in response to the natural androgens testosterone and dihydrotestosterone, the female sex steroid estradiol and the synthetic androgen R1881 (methyltrienolone). Testosterone and dihydrotestosterone at 1 microgram/ml enhance the acid phosphatase synthesis by a factor of 3.5, while a hundred-fold lower concentration of the synthetic androgen R1881 induces an almost five-fold increase in the expression of this enzyme. The stimulation by all androgens tested and estradiol was dose-dependent. The synthetic glucocorticoid triamcinolone acetonide does not modulate the prostatic acid phosphatase expression in LNCaP cells, neither alone nor in combination with R1881.     

The enzyme histochemistry of developing odontoblasts in cattle, pigs and horses

Synopsis The histochemical distribution of some hydrolytic and oxidative enzymes in developing odontoblasts and subodontoblasts in cattle, pigs and horses has been observed in cryostat sections of teeth that have been decalcified with neutral EDTA.Undifferentiated dental epithelium and immature odontoblasts of the bell stage tooth germ showed lower levels of enzymatic activity as compared with the well-developed tooth germ.When the dentine matrix began to form, the young odontoblasts appeared to have a significantly positive reaction for acid phosphatase, and gradually other enzymes developed an activity at the top of the cusp.Odontoblasts as well as subodontoblastic-rich cells showed strong enzymatic activities for hydrolytic and oxidative enzymes, that is, they were strongly reactive for alkaline and acid phosphatase and lactate and malate dehydrogenases, and moderately reactive for other oxidative enzyme systems.It is suggested that the subodontoblastic layer is concerned with the biosynthesis of dentinal matrix as well as with the odontoblasts themselves.