Kinetic intermediates of unfolding of dimeric prostatic phosphatase.

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 degrees 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.

Formation and detoxification of reactive oxygen species.

A model of reactive oxygen species metabolism is proposed as a laboratory exercise for students. The superoxide ion in this model is generated during the reaction of oxidation of xanthine, catalyzed by xanthine oxidase. The effect of catalase, superoxide dismutase, and allopurinol on superoxide ion generation and removal in this system is also determined, using cytochrome c as a probe.

From gene to protein: Prostatic acid phosphatase: Structure and expression of gene and protein*.

A set of classes for medical students is designed to reinforce an understanding of the basic laboratory methods of molecular biology and protein biochemistry in the context of a clinically important problem, prostate gland pathology. Students examine the gene coding for prostatic acid phosphatase and they assay expression of the gene in different lines of prostate cancer cell cultures (LNCaP and PC-3). The three-dimensional structure of the expressed protein is also investigated, in relation to its catalytic function. Students are encouraged to collect data for their experiments and to perform laboratory exercises on their own. The theory and practice should stimulate the students' discussion of various fields of biochemistry and molecular biology.

Equilibrium unfolding of dimeric human prostatic acid phosphatase involves an inactive monomeric intermediate.

Guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular weight, was investigated with activity measurements, size exclusion HPLC, tryptophan fluorescence, 1-anilinonaphtalene-8-sulfonate (ANS) binding and reactivity with 2-(4'-maleimidoanilino)naphthalene-6-sulfonate (MIANS). Equilibrium analysis was performed to shed light on the role of dimerization in the folding and stability of the catalytically active oligomeric protein. Unfolding was reversible, as verified by activity measurements and tryptophan fluorescence. The noncoincidence of the unfolding curves obtained by different techniques suggests the occurrence of a multiphasic process. The reaction of hPAP inactivation is accompanied by dissociation of the dimer into two monomers. The midpoint of this transition is at 0.65 M GdnHCl with 4.24+/-0.12 kcalmol(-1) free energy change. Binding of ANS to the inactive phosphatase monomer, especially remarkable in the region from 0.8 to 1.25M GdnHCl, suggests that the hydrophobic probe indicates exposition of the intersubunit hydrophobic surface and a loosening of the monomer's tertiary structure. Strong fluorescence of thiol group derivatives, the products of their reaction with MIANS, appears in a limited range of GdnHCl concentrations (1.2-1.6M). This shows that in the relaxed structure of the intermediate, the reagent is allowed to penetrate into the hydrophobic environment of the partially hidden thiol groups. The equilibrium unfolding reaction of hPAP, as monitored by tryptophan fluorescence, does not depend on the protein concentration and displays a single transition curve with a midpoint at 1.7 M GdnHCl and value of DeltaG(unf)(H(2)O)=3.38+/-0.08 kcalmol(-1) per monomer, a result implying that this transition is related to the conformational change of the earlier dissociated and already inactive subunit of the protein.

Application of a mixture of glycol polyethylenes for the preparation of microcorrosion casts--an observation.

Preparation of microcorrosion casts that can be used for observation in SEM is a laborious, time-consuming procedure. The authors paid particular attention to the process of dissection of the microcorrosion casts. This prompted the authors to reconstruct the plastic mass, produced by the firm Gurr (Great Britain) in the 1970s, which was used by them in previous research to immerse the cast in order to minimise the damage. By using easily obtainable polyethylene glycols, characterised by different physical and chemical features, in order to obtain smooth surface of the section, a low-toxic mixture was composed, which protected the microcorrosion casts sufficiently and did not interfere with the physical and chemical properties of the cast.