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The carbonate binding site on horse cytochrome c was mapped by comparing the yields of carboxydinitrophenyl-cytochromes c, each with a single carboxydinitrophenyl-substituted lysine residue per molecule, when the modification reaction was carried out in the presence and absence of carbonate. The site is located on the "left surface" of the protein and consists of lysine residues 72 and/or 73 as well as 86 and/or 87 (Carbonate Site). Although one of the binding sites for phosphate on cytochrome c (Phosphat Site I) is located near the carbonate site, the sites are distinctly different since carbonate does not displace bound phosphate, as monitored by 31P NMR. Furthermore, citrate interacts with Phosphate Site I with high affinity, whereas chloride, acetate, borate, and cacodylate have a much lower affinity for this site, if they bind to it at all. The affinity of phosphate for Phosphate Site I (KD = 2 X 10(-4) M) is at least 1 order of magnitude higher than it is for other sites of interaction.
Two groups of mares were exposed to an abrupt, artificial increase or a natural increase in daylength. In both groups, mean LH pulse frequency increased with time of year and was accompanied by a reciprocal decrease in LH pulse amplitude. A non-pulsatile pattern of LH secretion was observed in some mares sampled close to the day of ovulation. Maximum mean LH pulse frequency and the onset of the breeding season occurred earlier in those mares exposed to an abrupt artificial increase in daylength. In blood samples collected frequently, mean serum LH concentrations increased in relation to time of year. However, during 60 days before ovulation, when LH pulse frequency increased, mean daily serum LH values only increased on Day -3 before ovulation. The magnitude of the periovulatory LH rise was greater before the second than the first ovulation of the breeding season. These results support the hypothesis that, in the mare, a photoperiod-induced seasonal alteration in LH pulse frequency and/or amplitude may play a role in the onset of the breeding season.
Microsomes of human polymorphonuclear leukocytes (PMN) in the presence of 100 microM NADPH converted 0.6 microM leukotriene B4 (LTB4) to 20-OH-LTB4 (retention time = 18.0 min) and to two additional compounds designated I (retention time = 16.8 min) and II (retention time = 9.6 min) as analyzed by reverse-phase high performance liquid chromatography (HPLC). Compounds I and II were also formed from the reaction of 1.0 microM 20-OH-LTB4, PMN microsomes, and 100 microM NADPH; the identity of compound II was confirmed as 20-COOH-LTB4 by gas chromatography-mass spectrometry. Equine alcohol dehydrogenase in the presence of 100 microM NAD+ in 0.2 M glycine buffer (pH 10.0) converted 20-OH-LTB4 to 20-aldehyde (CHO) LTB4, which coeluted with compound I on reverse-phase HPLC. In the presence of 100 microM NADH in 50 mM potassium phosphate buffer (pH 6.5), equine alcohol dehydrogenase reduced both 20-CHO-LTB4 and compound I to 20-OH-LTB4, indicating the identity of compound I as 20-CHO-LTB4. Gas chromatography-mass spectrometry of trideuterated O-methyl-oxime trimethylsilyl ether methyl ester derivative of 3H-labeled compound I definitively established compound I as 20-CHO-LTB4. Addition of immune IgG to cytochrome P-450 reductase or 1.0 mM SKF-525A completely inhibited the formation of 20-CHO-LTB4 from 20-OH-LTB4, indicating that the reaction was catalyzed by a cytochrome P-450. LTB5 (3.0 microM), a known substrate for cytochrome P-450LTB and a competitive inhibitor of LTB4 omega-oxidation, completely inhibited the omega-oxidation of 1.5 microM 20-OH-LTB4 to 20-CHO-LTB4, indicating that the cytochrome P-450 was P-450LTB. Conversion of 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 by PMN microsomes was also dependent on NADPH and inhibited by antibody to cytochrome P-450 reductase, 1.0 mM SKF-525A, or 5.0 microM LTB5, indicating that this reaction was also catalyzed by cytochrome P-450LTB. These results identify the novel metabolite 20-CHO-LTB4 and indicate that cytochrome P-450LTB catalyzes three sequential omega-oxidations of LTB4 leading to the formation of 20-COOH-LTB4 via 20-OH-LTB4 and 20-CHO-LTB4 intermediates.
The relationship between daily mean FSH concentrations in serum and the pattern of FSH detected by frequent sampling for 12-h periods (samples every 15 min) was examined in five mares during the transition into the breeding season. The five mature anestrous mares were exposed to a natural increase in daylength. Blood samples were collected daily from February 1 until the first ovulation of the breeding season (April 14 +/- 3.7 days, Mean +/- SEM). Periods of frequent blood collection were performed every two weeks. Blood samples were obtained daily by jugular venipuncture or jugular cannula (frequent samples). Mean daily concentrations of FSH in serum determined by RIA decreased during seasonal transition. Patterns of FSH in serum detected by frequent sampling were pulsatile. FSH pulse amplitude decreased during seasonal transition, and the decrease in amplitude was associated with the decrease in mean serum FSH concentrations. This decrease in FSH pulse amplitude may reflect an involvement of a follicular product from developing follicles or a change in hypothalamic stimulation of pituitary FSH release.