Purification and characterization of human seminal plasma acid phosphatase

A 81-fold purification of human seminal plasma acid phosphatase was obtained by a three-step procedure, involving ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Homogeneity of the preparation during purification steps was tested by polyacrylamide gel electrophoresis and only one major band was obtained after the final step. The pH optimum for the activity of the purified enzyme was 5.6 and thermal stability was obtained even up to 40 degrees C. PNPP was the most specific synthetic substrate. The Km of purified seminal acid phosphatase towards PNPP was 1.5 X 10(-3) M. Among the metal ions tested, Hg+2 showed an I50 value of 4.2 X 10(-7) M. Studies with PCMB, PMSF and EDTA did not show any inhibition, whereas NaF and L(+)tartrate, at 1 mM concentration, inhibited the enzyme by 95% and 85%, respectively.     

Purification of prostatic acid phosphatase from human seminal plasma

Prostatic acid phosphatase has been isolated from human seminal plasma. The purification method utilizes gel filtration on Sephadex G100, ammonium sulfate precipitation and a series of chromatographical steps including concanavalin A Sepharose 4B, anion exchange and gel filtration chromatography. The final product appears homogenous when analyzed by gel filtration on Sephadex G100. It gives one major band on SDS polyacrylamide gels. The specific activity is similar to that obtained by other purification schemes. The yield of the method described above has allowed to set up a sensitive radioimmunoassay of prostatic acid phosphatase.     

Detection of prostatic tissue and seminal plasma peptides reacting with human seminal fluid acid phosphatase antibodies

IgG1 monoclonal antibody to purified seminal fluid phosphatase was raised by fusion of spleen cells from immunized mice with cell line Sp2/O-Ag 14 using simple method of screening for antiphosphatase antibody secreting clones. All molecular forms of catalytically active seminal fluid phosphatase and prostatic tissue phosphatase, resolved by chromatofocusing in pH gradient, react with this monoclonal antibody and with rabbit antiserum to purified seminal fluid phosphatase. Peptides of Mr 25,000 to 76,000 and of Mr 13,000 to 76,000 were adsorbed from the prostatic tissue extract and from seminal plasma on the monoclonal antibody-Sepharose column.     

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.     

Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min(-1) mg(-1). The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 +/- 1.5 kD. The K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.     

HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma

We recently reported that HIV-1 infection can be inhibited by innate antimicrobial components of human seminal plasma (SP). Conversely, naturally occurring peptidic fragments from the SP-derived prostatic acid phosphatase (PAP) have been reported to form amyloid fibrils called "SEVI" and enhance HIV-1 infection in vitro. In order to understand the biological consequence of this proviral effect, we extended these studies in the presence of human SP. PAP-derived peptides were agitated to form SEVI and incubated in the presence or absence of SP. While PAP-derived peptides and SEVI alone were proviral, the presence of 1% SP ablated their proviral activity in several different anti-HIV-1 assays. The anti-HIV-1 activity of SP was concentration dependent and was reduced following filtration. Supraphysiological concentrations of PAP peptides and SEVI incubated with diluted SP were degraded within hours, with SP exhibiting proteolytic activity at dilutions as high as 1:200. Sub-physiological concentrations of two prominent proteases of SP, prostate-specific antigen (PSA) and matriptase, could degrade physiological and supraphysiological concentrations of PAP peptides and SEVI. While human SP is a complex biological fluid, containing both antiviral and proviral factors, our results suggest that PAP peptides and SEVI may be subject to naturally occurring proteolytic components capable of reducing their proviral activity.     

Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana

Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase. LPA phosphatase gene has not been identified and characterized in plants so far. The BLAST search revealed that the At3g03520 is similar to phospholipase family, and distantly related to bacterial phosphatases. The conserved motif, (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases. In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity. These results suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50 kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5–7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K_m = 2.1 × 10⁻³ M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase.The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Purification and properties of alkaline phosphatase from boar seminal plasma

An alkaline phosphatase was purified from boar seminal plasma using adsorption to calcium phosphate gel, gel filtration, and ion-exchange chromatography. The preparation gave a single band on SDS polyacrylamide electrophoresis. The enzyme was a non-specific alkaline phosphatase that hydrolysed pyrophosphate slowly and had no phosphodiesterase activity. The pH optimum was 10 and the Km was approximately 0.2 mM with p-nitrophenyl phosphate as substrate. The enzyme was a zinc metalloenzyme as indicated by the loss of activity when treated with o-phenanthroline and the restoration of activity by zinc and magnesium ions. It also lost activity when treated with thiols. Molecular weight estimates from SDS polyacrylamide gel electrophoresis and gel filtration suggest that the enzyme is a tetramer of identical subunits, each of which has a molecular weight of 68,000.     

Purification of ram seminal plasma acid alpha-glucosidase

1. Ram seminal plasma alpha-glucosidase has been purified in order to increase our knowledge of this enzyme and of its role in epididymal physiology. 2. Since the enzyme behaved differently from other known acid alpha-glucosidases and was not affinity-adsorbed on dextran gels, another approach had to be used. 3. The final procedure included an ethanol precipitation step, sequential chromatography on hydroxylapatite and DEAE Sepharose CL-6B, isoelectric focusing in polyacrylamide gels and ultrafiltration. 4. The resulting purification factor of alpha-glucosidase was 9822 with an overall yield of 5%. 5. The purified material consisted of several isoforms with a mol. wt of 105,000 and isoelectric points varying between 4 and 5.     

Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6

Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein- coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L- (+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.     

The Saccharomyces cerevisiae PHM8 gene encodes a soluble magnesium-dependent lysophosphatidic acid phosphatase

Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.     

Characterization and localization of alkaline phosphatase in canine seminal plasma and gonadal tissues

Alkaline phosphatase (AP) is a useful indicator of the presence of the sperm-rich (2nd) fraction in the canine ejaculate. Two AP isoenzymes originating from separate genes have been identified in the dog: tissue nonspecific (TNS) and intestinal. Bone, liver, and corticosteroid-induced AP are different isoforms of the TNS and intestinal isoenzymes. Using gel electrophoresis and levamisole inhibition assays, it was determined that seminal plasma AP (SAP) is a unique isoform of canine TNS AP whose glycosylation is distinct from either of the TNS AP isoforms commonly found in canine serum. Using immunocytochemistry, SAP activity was localized to the epididymal and seminiferous tubular epithelium. The ability to distinguish SAP from bone AP, liver AP and corticosteroid-induced AP could be beneficial to the practitioner in determining the quality of a semen sample.     

Tartrate inhibition of prostatic acid phosphatase improves seminal fluid metabolite stability

Introduction

Human seminal fluid (hSF) has been suggested as a biofluid suitable to characterise male reproductive organ pathology with metabolomics. However, various enzymatic processes, including phosphorylcholine hydrolysis mediated by prostatic acid phosphatase (PAP), cause unwanted metabolite variation that may complicate metabolomic analysis of fresh hSF samples.

Objectives

To investigate the effects of PAP inhibition with tartrate.

Methods

Using NMR spectroscopy, the kinetics of phosphorylcholine to choline hydrolysis was characterized in hSF samples from three subjects at different temperatures and tartrate concentrations. Principal components analysis was used to characterise the effects of tartrate and temperature on personal differences in metabolite profiles. Potential effects of tartrate on RNA quantification were also determined.

Results

Metabolite profiles and the kinetics of phosphorylcholine degradation are reproducible in independent samples from three ostensibly normal subjects. Increasing concentrations of tartrate and refrigerated sample storage (279 K) resulted in greatly reduced reaction rates as judged by apparent rate constants. Multivariate statistical analysis showed that personal differences in metabolite profiles are not overshadowed by tartrate addition, which stabilises phosphorylcholine and choline concentrations. The tartrate signal also served as an internal concentration standard in the samples, allowing the determination of absolute metabolite concentrations in hSF. Furthermore, the presence of tartrate did not affect RNA expression analysis by qPCR.

Conclusion

Based on these results we recommend as standard protocol for the collection of hSF samples, that 10 mM tartrate are added immediately to samples, followed by sample storage/handling at 277 K until clinical processing within 6 h to remove/inactivate enzymes and isolate metabolite supernatant and other cellular fractions.

     

Prostatic acid phosphatase is an ectonucleotidase and suppresses pain by generating adenosine

Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5'-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain.     

Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid

Autotaxin (ATX) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA). ATX is secreted by adipose tissue and its expression is enhanced in obese/insulin-resistant individuals. Here, we analyzed the specific contribution of adipose-ATX to fat expansion associated with nutritional obesity and its consequences on plasma LPA levels. We established ATX(F/F)/aP2-Cre (FATX-KO) transgenic mice carrying a null ATX allele specifically in adipose tissue. FATX-KO mice and their control littermates were fed either a normal or a high-fat diet (HFD) (45% fat) for 13 weeks. FATX-KO mice showed a strong decrease (up to 90%) in ATX expression in white and brown adipose tissue, but not in other ATX-expressing organs. This was associated with a 38% reduction in plasma LPA levels. When fed an HFD, FATX-KO mice showed a higher fat mass and a higher adipocyte size than control mice although food intake was unchanged. This was associated with increased expression of peroxisome proliferator-activated receptor (PPAR)γ2 and of PPAR-sensitive genes (aP2, adiponectin, leptin, glut-1) in subcutaneous white adipose tissue, as well as in an increased tolerance to glucose. These results show that adipose-ATX is a negative regulator of fat mass expansion in response to an HFD and contributes to plasma LPA levels.