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Kinetic intermediates of unfolding of dimeric prostatic phosphatase

100%

Kuciel R., Mazurkiewicz A., Dudzik P.

Acta Biochimica Polonica

2007

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.

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Local effect of progesterone infusion into ovarian artery on activin A and inhibin alpha-subunit secretion during the middle luteal phase in gilts

80%

Wasowska B., Stefanczyk-Krzymowska S.

Polish Journal of Veterinary Sciences

2010

tom 13

nr 1

97-104

The present study was undertaken to elucidate whether an increased, but physiological, amount of progesterone (P4) supplied to the porcine corpus luteum affects luteal secretion of activin A and inhibin a-subunit (Inha) in freely moving gilts. On day 9 of the estrous cycle (EC), both ovarian arteries and both ovarian veins of gilts (n=5) were cannulated. Progesterone was infused into the right ovarian arteries in gilts on days 10, 11 and 12 of the EC at a rate adequate to its physiological retrograde transfer found during the middle luteal phase of the EC. The P4 infusion rate was 0.62 μg/min (day 10), 2x0.62 μg/min (day 11) and 3x0.62 μg/min (day 12). The left ovarian arteries were infused with saline (control). Blood samples were collected from both ovarian veins on days 10-12 of the EC before and after P4 or saline infusion. The mean plasma activin A level in the ovarian vein ipsilateral to the P4-infused ovary was higher (PcO.OOOl) on days 10-12 of the EC than this found in the contralateral ovarian vein. The level of activin A in the ovarian vein ipsilataral to the infusion of P4 was higher on days 11 (PcO.Ol) and 12 (P<0.0001) and tended to be higher (P<0.07) on day 10 of the EC than this in contralateral ovarian vein. The level of Inha in the ovarian vein ipsilateral to the P4-infused ovary on days 10-12 of the EC was not significantly different (P>0.05) than this found in the contralateral ovarian vein. The results of the present study indicate that a local elevation of P4 concentration in blood supplying the ovary during the middle luteal phase of the porcine EC affects ovarian secretion of activin A. The effect of P4 on the secretion of activin A suggested the existence of a short regulatory loop of a positive feedback between P4 being retrogradely transferred into the ovary and the secretion of this peptide.

Ograniczanie wyników

1 Acta Biochimica Polonica

1 Polish Journal of Veterinary Sciences

1 Dudzik P.

1 Kuciel R.

1 Mazurkiewicz A.

1 Stefanczyk-Krzymowska S.

1 Wasowska B.

1 2010

1 2007

Wyniki wyszukiwania

Kinetic intermediates of unfolding of dimeric prostatic phosphatase

Local effect of progesterone infusion into ovarian artery on activin A and inhibin alpha-subunit secretion during the middle luteal phase in gilts