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Acid phosphatase was purified to electrophoretic homogeneity from human normal lung and spleen and was characterized biochemically and immunologically in comparison with prostate acid phosphatase (PAP). The apparent MW of lung acid phosphatase (LAP) and spleen acid phosphatase (SAP) was 110,000 and 100,000, respectively, similar to that of PAP (100,000). All three enzymes exhibited similar electrophoretic mobility, optimal pH, substrate, and inhibitor specificity, except that PAP dephosphorylated profoundly the phosphate group from tyrosine phosphate in phosphoangiotensin (19,700 fmol/mg/min), whereas only marginal activities were detected for LAP and SAP (19 and 73 fmol/mg/min, respectively). Amino acid analysis revealed more similarity between SAP and LAP than PAP and LAP or PAP and SAP. An immunological cross-reactivity among these three acid phosphatases was detected by polyclonal and monoclonal antibodies raised against purified PAP, although unique epitopes were detected on the PAP molecule. This study provides data explaining why conventional biochemical methods are not specific for PAP measurement and why immunologic methods still detect other acid phosphatases, as observed in clinical laboratory assays. The data also suggest the possibility of using a new substrate or antibody reagent for a more specific assay for PAP.
Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.
Acid phosphatase is a ubiquitous lysosomal enzyme that hydrolyses organic phosphates at an acid pH. Although the postpuberteral prostatic epithelial cell contains a uniquely high concentration of acid phosphatase, cellular components of bone, spleen, kidney, liver, intestine, and blood also contain this enzyme. The discovery that prostatic carcinoma cells often retain a high concentration of acid phosphatase characteristic of the normal postpubertal gland led to the recognition of the first clinically useful tumor marker. Recognition that the serum of patients with prostatic malignancy frequently contains an increased concentration of this enzyme has resulted in persistent efforts to identify the source, to accurately quantitate the level of serum acid phosphatase, and to determine the clinical significance of those levels. A variety of enzymatic and immunologic techniques have been employed to measure acid phosphatase. In the past, various substrates and inhibitors were utilized to increase specificity and sensitivity. Emphasis has now shifted to the development of radioimmunoassay and counterimmunoelectrophoresis in an attempt to enhance those parameters. Judgment of their efficacy awaits further testing and evaluation. The clinical significance of normal and abnormal serum acid phosphatase is constantly being reevaluated. In order to maximize the value of laboratory measurements, the clinical and pathologic status of the patient, the techniques employed in obtaining and storing the blood sample and the procedures used in analysis must be known and considered. Traditionally, the serum prostatic acid phosphatase has been thought to originate in the prostatic cancer cell and has been used to stage the disease. Until recently, elevated serum values have been accepted as an indication of extraprostatic disease, and were thought to rule out lesions confined to the prostate. The elevation of acid phosphatase levels in patients with disseminated disease or the failure of elevated levels to return to normal with treatment have been assumed to indicate a poor prognosis. However, unequivocal documentation of the validity of these statements is not available. Newer immunologic techniques for measuring acid phosphatase may significantly alter our current concept of its role as a tumor marker.
Acid phosphatase of human seminal plasma is inhibited in presence of normal rabbit serum involving non-competitive mechanism whereas this character is normalized and activity is stabilized in presence of its rabbit antiserum. Kinetic studies on stabilization of acid phosphatase by antiserum were combined by L-tartrate, a specific inhibitor of prostatic acid phosphatase. L-tartrate, a specific inhibitor of prostatic acid phosphatase. L-tartrate inhibited the enzyme in presence and absence of normal and antiserum by competitive mechanism and percentage of inhibition of L-tartrate was involve in the binding with acid phosphatase antibodies. The kinetics of heat denaturation of acid phosphatase suggested that seminal acid phosphatase is more heat labile in presence and absence of normal serum than in presence of antiserum. The temperature of denaturation corresponding to half the initial activity(T 1/2) in presence of normal and antisera were 34 degrees and 66 degrees C respectively. NOn-linearity of Arrhenius plots for denaturation rates of the enzyme suggested various conformational states for denaturation yielding Ea varying between 20.08 and 5.03 Kcals at 25-65 degrees C.
Acid phosphatase activity in the blood serum is usually separated into tartrate-resistant and tartrate-refractory, which is reported as the prostatic acid phosphatase level. Human prostatic acid phosphatase crystals soaked in N-propyl-L-tartramate were used to collect x-ray diffraction data to 2.9 A resolution under cryogenic conditions. Positive difference electron density, corresponding to the inhibitor, was found. The quality of the electron density maps clearly shows the orientation of the carboxylate and N-propyl-substituted amide groups. The hydroxyl group attached to C3 forms two crucial hydrogen bonds with Arg-79 and His-257. Previous crystallographic studies compiled on the tartrate-rat prostatic acid phosphatase binary complex (Lindqvist, Y., Schneider, G., and Vihko, P. (1993) J. Biol. Chem. 268, 20744-20746) erroneously positioned D-tartrate into the active site. Modeling studies have shown that the C3 hydroxyl group on the D(-)-stereoisomer of tartrate, which does not significantly inhibit prostatic acid phosphatase, does not form strong hydrogen bonds with Arg-79 or His-257. The structure of human prostatic acid phosphatase, noncovalently bound in N-propyl-L-tartramate, is used to develop inhibitors with higher specificity and potency than L(+)-tartrate.
In 25 patients with carcinoma of the prostate (CaP) T3 and in a comparative group of 18 patients with BPH the serum enzymes of AP, tartrate labile AP, LDH, and iso-LDH were investigated simultaneously in basal conditions and after standardized transrectal prostatic biopsy. AP, PAP as well as LDH were shown to be of small diagnostic aid. The reaction of serum enzyme levels following the standardized prostatic biopsy was the same in both CaP and BPH patients. In studying LDH-isoenzymes, we found that the third fraction was elevated in almost all patients. This change is apparently not of prostatic origin, and we could not attribute it to the concomitant diseases found in some patients.
Serial levels of PAP and AcP activity were compared for their relative values in monitoring 57 early and 33 advanced prostate cancer patients. Several findings regarding the patients' disease status and the enzyme levels have been observed that may be beneficial to therapeutic management of these patients. They are: [1] an elevated PAP activity in disease recurrence and disease progression generally precedes an elevated AcP activity, and thus represents a more sensitive index for patients with early and advanced disease; [2] serial mean levels of PAP activity greater than the mean + 3 SD are more predictive for disease recurrence and progression than are those of AcP activity in both groups of patients; [3] PAP activity is a more sensitive monitor for changes in objective treatment response than is AcP activity; and [4] PAP is more specific than AcP for prostate, thus offering a more reliable marker to identify metastasis of unknown origin, or to confirm metastasis derived from a primary prostate tumor that may have been suggested by other non-prostate-specific marker[s]. In addition, data suggest a favorable prognosis for patients receiving therapy as inferred by a serial mean of PAP activity that is less than mean + 3 SD.
Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study, we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 microM, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.
Acid phosphatase in S. cerevisiae exists as an enzymatically active, cell wall associated form and as an enzymatically inactive, probably membrane-bound form (Schweingruber and Schweingruber, in press). Orthophosphate dependent and independent regulation determines the level of acid phosphatase activity. To deduce the regulation mechanisms we purified and quantified active and inactive acid phosphatase from cells grown under different physiological conditions and displaying variable levels of enzyme activity. Orthophosphate dependent regulation does not include significant changes in the amount of total (active and inactive) acid phosphatase protein synthesized. Under the experimental conditions chosen increased activity is achieved by preferential synthesis of the active form and by increasing the specific activity of the active enzyme. Orthophosphate independent regulation seems to occur by similar mechanisms.
Yellow-brown bodies were observed in the sinusoids of lymph node and histiocytes. The authors confirmed immunohistochemical reactivity of lysozyme, alpha-1-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid phosphatase in non-phagocytic and phagocytic histiocytes which contained yellow-brown bodies. Phagocytic histiocytes (histiocytes with yellow-brown bodies) were not reacted with lysozyme, alpha-1-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid phosphatase. On the other hand, non-phagocytic histiocytes were reacted with lysozyme, alpha-1-antichymotrypsin, S-100 protein, alkaline phosphatase, and acid phosphatase.
Acid phosphatase has been characterized and quantified in human suction blister fluid, interstitial fluid and serum. The acid phosphatases of suction blister fluid and serum showed differences in their pH activity curves, Michaelis-Menton constants, heat stabilities and sensitivities to inhibition by tartrate and fluoride. The behaviour of the interstitial fluid enzyme was intermediate between these two. The levels of activity in blister fluid and serum were very similar, both being more than twice that of interstitial fluid. These results suggest that the acid phosphatase activity in suction blister fluid is derived largely from the overlying epidermis rather than from serum.
Acid phosphatases (orthophosphoric-monoester phosphohydrolases (acid optimum), EC 3.1.3.2) of low and high molecular weight were separated by Sephadex G-75 filtration from extracts of rat brain, liver and kidney. The proportion of each phosphatase in the extract depends critically on the method employed for homogenate preparation, and no interconversion between high and low molecular weight forms was detected. In extracts obtained from subcellular organelles only high-molecular-weight acid phosphatase was detected, which is of lysosomal origin. Low-molecular-weight acid -phosphatase is restricted to the cell sap. Low- and high-molecular-weight acid phosphatases were characterized by their elution volumes, specific inhibition and activity with two substrates. It is suggested that the distribution pattern found om rat tissues could be common to all eukaryotic cells.
Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.
Acid phosphatases (APs) are a family of enzymes that are widespread in nature, and can be found in many animal and plant species. Mystery surrounds the precise functional role of these molecular facilitators, despite much research. Yet, paradoxically, human APs have had considerable impact as tools of clinical investigation and intervention. One particular example is tartrate resistant acid phosphatase, which is detected in the serum in raised amounts accompanying pathological bone resorption. This article seeks to explore the identity and diversity of APs, and to demonstrate the relation between APs, human disease, and clinical diagnosis.