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R Kuciel

Publications and source records attributed to R Kuciel.

15 recordsLinked to original sources
PubMed2000

Crystal structure of human prostatic acid phosphatase .

BACKGROUND: Prostatic acid phosphatase (hPAP) is a major product of the human prostate gland, yet its physiological substrate remains unknown. METHODS: Human PAP, purified from semen, was crystallized using polyethylene glycol as the precipitant and its crystal structure was determined using X-ray diffraction. The structure was refined at 3.1 A resolution to R = 16% and R(free) = 27%. RESULTS: The structure of hPAP is similar to that of other known histidine phosphatases, and the positions of its catalytic residues are conserved. N-linked carbohydrates are present at each of the possible glycosylation sites. It appears that high-mannose chains are attached to Asn 62 and Asp 301, while complex chains are at Asn 188. CONCLUSIONS: The similarity of the three-dimensional structures of rat PAP and human PAP indicates that the mechanistic analyses of the catalytic mechanism proposed for the rat enzyme should be extended to the human enzyme without reservations. The crystallographic data allowed the correlation of attachment sites of N-linked carbohydrate chains with a given carbohydrate type. The carbohydrates of the protein produced in the prostate cells and in the baculovirus expression system appear to differ at the site of complex carbohydrates attachment.

Acid Phosphatase↗
PubMed1997

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

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.

Acid Phosphatase↗
PubMed1997

Prostatic acid phosphatase: structural aspects of inhibition by L-(+)-tartrate ions.

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.

Acid Phosphatase↗
PubMed1996

The folding intermediate of reversibly denatured human prostatic acid phosphatase.

Human prostatic acid phosphatase (hPAP) [EC 3.1.3.2], a homodimer of ca. 50 kDa subunit molecular weight, shows reversible denaturation in 6 M urea at pH 2.5. Rapid dilution of the denatured enzyme allowed partial renaturation of hPAP, as measured by enzyme activity, to a level which depended on the composition of the dilution solution employed and time of the reaction. The renaturation reaction of hPAP was examined using spectral analysis (circular dichroism and fluorometry), fast size-exclusion chromatography and proteolysis by trypsin. The observed results are in agreement with the concentration-dependent kinetics of hPAP reactivation, assuming that the reconstitution of the active enzyme requires the association of subunits in dimeric form. Moreover, it suggests formation of an inactive intermediate during refolding of the denatured PAP. A mechanism of renaturation of the active enzyme from denatured PAP is proposed.

Acid Phosphatase↗
PubMed1994

Human prostatic acid phosphatase: selected properties and practical applications.

Human prostatic acid phosphatase (EC 3.1.3.2) is a non-specific phosphomonoesterase, synthetized and secreted into seminal plasma under androgenic control. The enzyme is a dimer of molecular weight around 100 kDa. Gene coding this protein is localized on chromosome 3. Since many years prostatic phosphatase has been used as a marker of diagnosis and therapy control of cancer of the prostate gland. The biological role of this enzyme, however, remains unknown and needs further exploration.

Acid Phosphatase↗
PubMed1993

Fluorometric analysis of native, urea-denatured and refolded human prostatic acid phosphatase.

Human prostatic acid phosphatase (EC 3.1.3.2) was denatured in 6 M urea at pH 2.5, but was refolded by dilution at pH 7.0, as demonstrated by the recovery of nearly complete enzyme activity and dimeric structure. The conformational changes among the native, denatured and refolded states were monitored by means of steady-state and nanosecond pulse fluorometry of tryptophan residues of the enzyme. The relative quantum yield of the fluorescence was highest in the native enzyme and lowest in the denatured one, and was intermediate in the refolded enzyme, although the emission peak was reproducible after refolding. The observed decay curves of tryptophan fluorescence of the native, denatured and refolded states were analyzed by decay functions of three lifetimes. The fluorescence lifetimes of the refolded enzyme were shorter than those of the native one. The fluorescence of the denatured enzyme decayed much faster than that of the other forms. The fluorescence excitation spectra revealed that the excitation energy of phenylalanine was transferred to tryptophan(s) in the native and refolded forms, but not in the denatured form. The efficiency of the energy transfer was higher in the native enzyme than in the refolded one. It was found by excitation polarization spectra that the freedom of internal motion of tryptophans was greater in the refolded enzyme than in the native enzyme. In the denatured enzyme the polarization anisotropies were very low. These results indicate that the higher structure with respect to tryptophans of the refolded enzyme is delicately but definitely different from that of the native enzyme and that local conformation of the active center is recovered upon refolding.

Acid Phosphatase↗
PubMed1992

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.

Acid Phosphatase↗
PubMed1990

Is the subunit of prostatic phosphatase active? Reversible denaturation of prostatic acid phosphatase.

Prostatic acid phosphatase [E.C. 3.1.3.2.] is a dimeric protein consisting of two identical subunits. This enzyme was denatured in 6 M urea solution at pH 2.5, and kinetical analysis of reactivation by dilution was performed. At low protein concentrations a second-order kinetics for reactivation of phosphatase, with rate constant 8.3 m M-1sec-1, was observed. At higher protein concentrations the reactivation obeyed first-order kinetics. These results seem to exclude the possibility that subunits of the prostatic phosphatase are catalytically active and suggest that the association of two monomers is necessary for full activity.

Acid Phosphatase↗
PubMed1988

Immunoreactivity of proteolytic degradation products of human prostatic acid phosphatase.

Prostatic acid phosphatase (EC 3.1.3.2) was fragmented by trypsin and papain in the presence of sodium dodecyl sulphate. Trypsin-catalysed cleavage gave a peptide of 33 kDa which was subsequently trimmed to 18 kDa, 15 kDa and 13 kDa peptides. Even the small tryptic fragments reacted with antiphosphatase antibodies from rabbit serum and with monoclonal antibody mAb-14. Papain treatment under these conditions resulted in the release of a 40 kDa peptide which was gradually reduced to a 18 kDa peptide. The monoclonal antibody mAb-14 to the prostatic phosphatase was bound exclusively to the 50 kDa subunit of the phosphatase and to the 40 kDa peptide. The results suggest that the monoclonal antibody mAb-14 binding site represents a "local" sequence rather than a "conformational" one and does not require an extensive tertiary folding of the antigen molecule.

Acid Phosphatase↗
PubMed1988

Characterization of anti-prostatic acid phosphatase monoclonal antibody and its medical significance.

Monoclonal antibody to purified prostatic acid phosphatase from seminal plasma was produced by fusion of spleen cells from immunized mice with the Sp2/O-Ag 14 cell line. This hybridoma-derived antibody, designated MAb-14, was classified as IgG1 immunoglobulin. The apparent affinity constant of phosphatase.MAb-14 complex formation calculated by using the Langmuir isotherm is 2.4 x 10(-8) M. The molecular weight of the complex formed under the condition of antibody excess was found to be 350K, which suggests that 2 molecules of prostatic phosphatase bind to 1 molecule of the antibody. The MAb-14 antibody bound to phosphatase had a negligible effect on the catalytic activity of the enzyme. All isoenzymatic forms of catalytically active prostatic phosphatase, resolved by isoelectric focusing or by chromatofocusing in different pH gradients, reacted with the monoclonal antibody. Several peptides of Mr 25K to 76K and of 13K to 76K were adsorbed from the prostatic tissue extract and from seminal fluid, respectively, on an MAb-14-Sepharose column. The MAb-14 monoclonal antibody was applied to the immunohistochemical investigation of prostatic phosphatase distribution in normal human prostate gland, in nodular hyperplasia, and in adenocarcinoma of the prostate. Immunostaining was observed in prostatic secretory epithelium, within the luminal content of prostatic glands, and in the neighborhood of prostatic cancer cells. Metastatic prostatic carcinoma was also strongly immunoreactive with the antibody. There was no cross-reactivity with leukocytes, kidney, liver, pancreas, spleen, breast, stomach mucosal, and colon tissues.

Acid Phosphatase↗
PubMed1986

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.

Acid Phosphatase↗
PubMed1985

Phosphotyrosine as a substrate of acid and alkaline phosphatases.

A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.

Acid Phosphatase↗
PubMed1975

Preparation and characterisation of ribonuclease from human hypertrophic prostate gland (RNAase P2).

An endo-type, cyclising, 3'-phosphate-forming rebonuclease was purified to homogeneity from a water/Tween 80 extract of human hypertrophic prostate gland. The enzyme is acid- and heat- resistant and is optimally active at pH 7.0, 0.1 M NaCl. Molecular weight determined by gel filtration on Sephadex G-75 and sucrose density gradient centrifugation gave a mean value of 15 000. The prostatic ribonuclease is inhibited by Cu2+, bromoacetate and photooxidation in the presence of methylene blue. Other divalent ions, EDTA and p-chloromercuribenzoate have no influence on the enzymic activity. Prostatic RNase resembles RNase A in that it preferentially cleaves linkages in RNA after pyrimidine nucleotides to produce oligonucleotides terminated in cyclic 2',3' phosphate. The enzyme is inactive with poly(A) - poly(U) as substrate. Poly(U) is hydrolyzed four times as fast as poly(C), and 1.2 times as fast as RNA.

Cations, Divalent↗
PubMedDate not supplied

Enzyme markers in human prostatic carcinoma.

In an effort to develop a sensitive and specific method for detecting human prostatic cancer at early stages, we have studied the isoenzyme patterns of acid phosphatase in patients' sera as well as in benign hypertrophic and cancerous prostatic tissues using isoelectric focusing techniques. At least eight acid phosphatase isoenzymes at pI 4.1-5.5 could be observed. The sera with highly elevated acid phosphatase activity generally contained more isoenzymes with pI values of 4.5-5.0. The purified acid phosphatase isolated from benign hypertrophic and malignant prostatic tissues showed no qualitative difference in isoenzyme patterns although quantitative variations were observed. Malignant tissue contained more isoenzymes with pI values of 4.5-4.8. Patients' sera were found to contain isoenzymes of prostate origin. We have also investigated serum ribonuclease (RNase) activity in patients with prostatic cancer. The serum RNase activity of patients was significantly elevated. No significant correlation was observed between serum acid phosphatase and RNase activity. In some instances, where acid phosphatase activity was in the normal range, RNase activity was elevated. These data suggest that simultaneous measurements of RNase and acid phosphatase activities may be of value in the diagnosis of prostatic cancer. The purified RNase has been isolated from human prostatic tissue and its immunologic properties are being studied.

Acid Phosphatase↗