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1

Prostatic acid phosphatase: structural aspects of inhibition by L-[plus]-tartrate ions

100%
Lovelace L., Lewinski K., Jakob C. G., Kuciel R., Ostrowski W., Lebioda L.
Acta Biochimica Polonica
|
1997
|
tom 44
|
nr 4
The crystal structure of the complex between rat-prostatic acid phosphatase (PAP) and L-(+)-tartrate (Lindqvist et al., J. Biol. Chem., 1993, 268, 20744-20746) contains the model of the ligand with incorrect chirality. We report here the correct model and discuss the relation between this model and the model of the inhibitory complexes between PAP and oxy-anions.
2

Continuous assay for acid phosphatase using 1-naphthyl phosphate as a substrate

88%
Luchter-Wasylewska E.
Acta Biochimica Polonica
|
1997
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tom 44
|
nr 4
The described continuous acid phosphatase assay is based on kinetics of the release of 1-naphthol in the course of the enzyme-catalyzed hydrolysis of 1-naphthyl phosphate, measured at 320 nm in aqueous solution and at 322 nm in sodium-bis(2-ethylhexyl)sulfosuccinate isooctane-water reverse micelles in a broad pH range (1.0-8.2). The method allows precise determination of the initial rate of the reaction and therefore may be used in the steady-state and pre-steady-state studies on the phosphatase-catalyzed reaction. The kinetic parameters (Km and kcat) for human prostatic acid phosphatase in aqueous solution and in reverse micelles, at pH 3.8, 4.5 and 5.7, by the proposed 1-naphthyl phosphate assay have been determined.
3

Molten globule as an intermediate on the human prostatic phosphatase folding pathway

88%
Kuciel R., Mazurkiewicz A.
Acta Biochimica Polonica
|
1997
|
tom 44
|
nr 4
Human prostatic acid phosphatase (hPAP, EC.3.1.3.2), a secretory homodimeric protein was denatured in 6 M urea, pH 2.5, and refolded by dilution at pH 7.2 with recovery of the enzymatic activity and dimeric structure. Circular dichroism, intrinsic fluorescence and chromatographic analysis of renaturating protein suggested that the kinetic intermediate of the hPAP folding is a monomer which displays a molten globule state (R. Kuciel, A. Mazurkiewicz & W.S. Ostrowski, 1996, Int. J. Biol. Macromol. 18, 167-175). To confirm these data experiments were performed to estimate the interaction of the renaturating protein with dyes and amphipathic lipid structures. Increased binding of the hydrophobic probe 1-anilinonaphthalene-8-sulfonate and Congo Red to the refolding enzyme supported the existence of molten globule state with the relaxed beta-structure in the renaturating protein. Presence of liposomes, included in the renaturation mixture as a model of acid phospholipid, resulted in perturbations of the human PAP refolding process. Some folding intermediates were bound to phosphatidylserine liposomes or, alternatively, water soluble, inactive aggregates were formed.
4

The effect of dihydrotestosterone on transcription of prostatic acid phosphatase mRNA in human hyperplastic gland

75%
Dulinska J., Laidler P., Ostrowski W. S., Mrozicki S., Galka M.
Acta Biochimica Polonica
|
1997
|
tom 44
|
nr 4
The effect of 5alpha-dihydrotestosterone (DHT) on the level of human prostatic acid phosphatase (hPAP) mRNA was studied using tissue slices from various benign prostatic hyperplastic glands. The absence of DHT in the incubation medium led to a gradual, significant decrease of the hPAP mRNA level. Addition of the hormone induced hPAP mRNA in a time- and dose-dependent manner. The maximal 2-4-fold induction by 10(-9) M DHT was observed after 3-5 h of incubation, and then the hPAP mRNA level was 6-20-fold higher than that in a parallel sample incubated without DHT. The results suggest that DHT is necessary to sustain the expression of hPAP in hyperplastic prostates.
5

High sequence homology between protein tyrosine acid phosphatase from boar seminal vesicles and human prostatic acid phosphatase

63%
Wysocki P., Plucienniczak G., Strzezek J.
Acta Biochimica Polonica
|
2009
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tom 56
|
nr 3
481-486
Boar seminal vesicle protein tyrosine acid phosphatase (PTAP) and human prostatic acid phosphatase (PAP) show high affinity for protein phosphotyrosine residues. The physico-chemical and kinetic properties of the boar and human enzymes are different. The main objective of this study was to establish the nucleotide sequence of cDNA encoding boar PTAP and compare it with that of human PAP cDNA. Also, the amino-acid sequence of boar PTAP was compared with the sequence of human PAP. PTAP was isolated from boar seminal vesicle fluid and sequenced. cDNA to boar seminal vesicle RNA was synthesized, amplified by PCR, cloned in E. coli and sequenced. The obtained N-terminal amino-acid sequence of boar PTAP showed 92% identity with the N-terminal amino-acid sequence of human PAP. The determined sequence of a 354 bp nucleotide fragment (GenBank accession number: GQ184596) showed 90% identity with the corresponding sequence of human PAP. On the basis of this sequence a 118 amino acid fragment of boar PTAP was predicted. This fragment showed 89% identity with the corresponding fragment of human PAP and had a similar hydropathy profile. The compared sequences differ in terms of their isoelectric points and amino-acid composition. This may explain the differences in substrate specificity and inhibitor resistance of boar PTAP and human PAP.
6

Kinetic intermediates of unfolding of dimeric prostatic phosphatase

63%
Kuciel R., Mazurkiewicz A., Dudzik P.
Acta Biochimica Polonica
|
2007
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tom 54
|
nr 2
Kinetics of guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular mass was investigated with enzyme activity measurements, capacity for binding an external hydrophobic probe, 1-anilinonaphtalene-8-sulfonate (ANS), accessibility of thiols to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS) and ability to bind Congo red dye. Kinetic analysis was performed to describe a possible mechanism of hPAP unfolding and dissociation that leads to generation of an inactive monomeric intermediate that resembles, in solution of 1.25 M GdnHCl pH 7.5, at 20°C, in equilibrium, a molten globule state. The reaction of hPAP inactivation in 1.25 M GdnHCl followed first order kinetics with the reaction rate constant 0.0715 ± 0.0024 min-1. The rate constants of similar range were found for the pseudo-first-order reactions of ANS and Congo red binding: 0.0366 ± 0.0018 min-1 and 0.0409 ± 0.0052 min-1, respectively. Free thiol groups, inaccessible in the native protein, were gradually becoming, with the progress of unfolding, exposed for the reactions with DTNB and MIANS, with the pseudo-first-order reaction rate constants 0.327 ± 0.014 min-1 and 0.216 ± 0.010 min-1, respectively. The data indicated that in the course of hPAP denaturation exposure of thiol groups to reagents took place faster than the enzyme inactivation and exposure of the protein hydrophobic surface. This suggested the existence of a catalytically active, partially unfolded, but probably dimeric kinetic intermediate in the process of hPAP unfolding. On the other hand, the protein inactivation was accompanied by exposure of a hydrophobic, ANS-binding surface, and with an increased capacity to bind Congo red. Together with previous studies these results suggest that the stability of the catalytically active conformation of the enzyme depends mainly on the dimeric structure of the native hPAP.
7

Comparative analysis of prostatic acid phosphatase and prostate-specific antigen mRNA levels in hyperplastic prostate stimulated with steroid hormones and growth factors

63%
Dulinska J., Laidler P., Labedz M.
Acta Biochimica Polonica
|
2002
|
tom 49
|
nr 2
Prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) are the mark­ers of human prostatic gland. However, it is still not completely understood if and how, steroid hormones and growth factors affect their expression and metabolism in the respect to the major pathologies of the gland. Appropriate studies were carried out on histopathologically diagnosed benign prostatic hyperplasia — BPH (n = 42) using tissue slices and cells derived from them. They were incubated with steroid hormones: 5-α-dihydrotestosterone (DHT), estradiol (E) and growth factors: epidermal growth factor (EGF), basic fibroblastic growth factor (bFGF) under culture conditions for up to 24 hours. 32P-labelled specific oligonucleotide probes were used to analyze total RNA isolated from each sample for the presence of PAP and PSA mRNAs. DHT, E, bFGF, EGF or both DHT + bFGF and DHT + EGF increased PAP and PSA mRNA levels in a time- and dose-dependent manner. The highest and statistically sig­nificant increase (P < 0.001) for PAP mRNA was observed when DHT + bFGF were present in the medium while for PSA mRNA if DHT + EGF were added to the medium. Slow but constant decrease of PAP and PSA mRNA levels was observed in the absence of each of these factors in the incubation medium. The results suggest that early expression of PSA and PAP genes and/or their mRNA stability strongly depend on DHT while differ in their response to EGF and bFGF.
8

Covalent modification and site-directed mutagenesis of an active site tryptophan of human prostatic acid phosphatase

63%
Zhang Z., Ostanin K., Van Etten R. L.
Acta Biochimica Polonica
|
1997
|
tom 44
|
nr 4
Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.
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