Effect of nucleoside triphosphates on cyclic nucleotide phosphodiesterase: role of protein modulators

The effects of nucleoside triphosphates (ATP and GTP) on phosphodiesterase (PDE) of brain and outer segments of the retina enriched or devoid of protein modulators were studied. In the case of retinal outer segment PDE the enzyme activity was considerably inhibited by both nucleosides only when the enzyme was separated from the inhibitor. In case of brain PDE, on the contrary, the effect of the nucleosides was much more pronounced in the enzyme preparation coupled with the protein activator, calmodulin. The latter when added to brain PDE devoid of the activator in the presence of ATP and GTP considerably reduced the enzyme activity. An addition of the inhibitor simultaneously with GTP to the purified PDE of outer segments increased the PDE activity. The constants for the inhibition of brain PDE coupled with calmodulin and retinal outer segment PDE separated from the inhibitor by ATP and GTP were determined.     

Progesterone metabolism in T47Dco human breast cancer cells--II. Intracellular metabolic path of progesterone and synthetic progestins

We show here that progesterone added to the medium of proliferating T47Dco human breast cancer cells is metabolized with a half life of 2-4h. The final metabolic product, 5 alpha-pregnan-3 beta,6 alpha-diol-20-one, (P-metabolite) is released into the medium. This structure suggested that the intracellular metabolism of progesterone involves the enzymes 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase, and 6 alpha-hydroxylase. To investigate this pathway, the cells were incubated with a variety of potential substrates. In addition to progesterone, only precursors with the 5 alpha-configuration served as substrates for the enzymes leading to P-metabolite formation. Some precursors with a 5 beta-configuration were also metabolized by T47Dco cells. This metabolism reflected activity by either 3 beta-hydroxysteroid dehydrogenase and/or 6 alpha-hydroxylase but, in contrast to progesterone metabolism, the rates were different and the products were often mixtures. In T47Dco and MCF-7 human breast tumor cells, the reduction at C-3 followed by 6 alpha-hydroxylation, appear to be the major, and possibly only, route of progesterone metabolism. In contrast, preliminary data suggest that in normal human breast epithelial cells, this is not an exclusive route. Androgens are partially subject to the same metabolic enzymes, but synthetic progestins are not metabolized by T47Dco during an 18 h incubation.     

Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.     

Susceptibility of human and murine Chlamydia trachomatis serovars to granulocyte- and epithelium-derived antimicrobial peptides.

Four antimicrobial peptides, protegrin-1, RTD-1, cryptdin-4, and indolicidin, were tested for their ability to inhibit the in vitro growth of Chlamydia trachomatis serovars E, L2, and mouse pneumonitis (MoPn). In general, protegrin-1 was found to have the strongest anti-chlamydial activity. Overall, of the three serovars tested, L2 was the most susceptible while MoPn was the most resistant to these peptides.     

Activity of cAMP-dependent protein kinases and cAMP-binding proteins from rat renal cytosol upon dehydration

The activity of cAMP-dependent protein kinases, cAMP binding and the spectrum of cAMP-binding proteins in renal papillary cytosol of intact rats and of rats kept on a water-deprived diet for 24 hours were investigated. It was found that the stimulation of protein kinases by 10(-6) M cAMP in the experimental group was significantly higher than in the control one. On DEAE-cellulose chromatography, the position of peaks of the specific cAMP binding corresponded to those of the regulatory cAMP-dependent protein kinases type I and II. Under these conditions, more than 80% of the binding activity in intact animals was localized in peak II, whereas in rats kept on a water-deprived diet over 60% of the binding activity was localized in peak I. The total binding activity of cytosol in experimental animals remained unchanged is compared to intact rats. It is suggested that in renal papilla dehydration is accompanied by the induction of synthesis of regulatory subunits of cAMP-dependent protein kinase type I.