pH-dependent behaviour of erythrocyte membrane elevations.

Freeze-etch electron microscopy of human erythrocytes from heparinized normal blood reveals membrane elevations. These elevations disappear at low pH values and reappear under normal pH conditions in vitro, both in the absence and the presence of plasma. This pH-dependent behaviour is discussed as an intrinsic membrane event.     

Pulsatile patterns of gonadotropins and ovarian steroids during estrus and pseudopregnancy in the rabbit

Consecutive daily plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17 beta (E2), progesterone (P4) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) were monitored in estrous rabbits and in these same doses during pseudopregnancy (PSP); these daily hormone levels, as well as the immediate post-coital changes in gonadotropin secretion, were similar to those in previous reports. To examine the pulsatile patterns of the gonadotropins and ovarian steroids, sequential, 10-min plasma samples were collected for 6 h from estrous does and on Days 3, 10, and 17 of PSP. All five hormones were measured in the serial samples from estrous and PSP Day 10 does; LH and FSH only were assayed in the remaining sequential samples. The amplitude and frequency of FSH pulses did not differ between any of these stages. In marked contrast, LH pulse amplitudes, and even pulse frequencies in Day 17 does, were profoundly increased during PSP above those in estrous does. Progestin secretions, both P4 and 20 alpha-OHP, also were sharply elevated in PSP Day 10 does as compared with those in estrous rabbits; the pulse amplitudes of both progestins were severalfold higher during PSP. P4 pulse frequencies were also increased at this time. Conversely, the parameters of E2 secretion did not differ between estrous and PSP Day 10 animals. In PSP Day 10 does, high amplitude pulses of both P4 and 20 alpha-OHP occurred simultaneously with high amplitude LH pulses. Simultaneous E2 and P4 pulses were evident in these same sequential plasma samples, and this E2-P4 pulse association was greater than that of 20 alpha-OHP pulses with E2 pulses. Our findings failed to identify conclusively the trophic stimulus for the progestin pulse patterns, but the mechanism may involve the coordinated action of LH and E2. The results do demonstrate that each gonadotropin and ovarian steroid is secreted in a pulsatile manner in both estrous and pseudopregnant rabbits. There are altered profiles in LH and progestin pulses, without major changes in FSH and E2 patterns, between the stages of estrus and PSP. The causes and consequences of these divergent endocrine shifts cannot be deduced from these data.     

An enzyme kinetics and 19F nuclear magnetic resonance study of selectively trifluoroacetylated cytochrome c derivatives.

The reaction of cytochrome c with ethyl thioltrifluoroacetate was carried out under conditions which led to the selective trifluoroacetylation of a small number of the 19 lysines. The mixture of derivatives was separated by ion-exchange chromatography and four different derivatives with well-resolved 19F nuclear magnetic resonance (NMR) spectra were obtained. Peptide mapping techniques indicated that one of these derivatives contained a single trifluoroacetyl group at lysine 22, and another derivative was singly labeled at lysine 25. The trifluoroacetylated lysine 22 derivative was fully active toward both succinate-cytochrome c reductase (EC 1.3.99.1) and cytochrome oxidase (EC 1.9.3.1) white the trifluoroacetylated lysine 25 derivative was fully active toward the reductase, but had a threefold greater Michaelis constant in the cytochrome oxidase reactin. This supports the hypothesis that the cytochrome oxidase binding site is located in the heme cervice region, and that Lys-25 is important in the binding. 19FNMR spectra of the cytochrome c derivatives bound to phospholipid vesicles were obtained. The reasonably narrow line widths (35-65 Hz) and good sensitivity of the trifluoroacetyl resonances indicated that they might be useful probes for the interaction of cytochrome c with intact mitochondria.     

Isolation and structure of the sex-pheromone of the moth, Nudaurelia cytherea cytherea

The sex-pheromone of Nudaurelia cytherea cytherea has been isolated from the terminal segments of the female moth by solvent extraction, steam distillation, and column and gas-liquid chromatography. The structure was determined by infrared, nuclear magnetic resonance, and mass spectrometry as cis-dec-5-en-1-yl 3-methylbutanoate (isovalerate). The synthetic ester showed activity in field tests.     

Determinants of human glucokinase activation and implications for small molecule allosteric control

Glucokinase (GK) is an enzyme that catalyzes the ATP-dependent phosphorylation of glucose to form glucose-6-phosphate, and it is a tightly regulated checkpoint in glucose homeostasis. GK is known to undergo substantial conformational changes upon glucose binding. The monomeric enzyme possesses a highly exotic kinetic activity profile with an unusual sigmoidal dependence on glucose concentration. In this interdisciplinary study, which draws on small angle X-ray scattering (SAXS) integrated with 250?ns of atomistic molecular dynamics (MD) simulations and experimental glucose binding thermodynamics, we reveal that the critical regulation of this glucose sensor is due to a solvent controlled “switch”. We demonstrate that the “solvent switch” is driven by specific protein structural dynamics, which leads to an enzyme structure that has a much more favorable solvation energy than most of the protein ensemble. These findings uncover the physical workings of an agile and flexible protein scaffold, which derives its long-range allosteric control through specific regions with favorable solvation energy. The physiological framework presented herein provides insights that have direct implications for the design of small molecule GK activators as anti-diabetes therapeutics as well as for understanding how proteins can be designed to have built-in regulatory functions via solvation energy dynamics.     

Amniotic fluid and decidual prolactin during pregnancy in rhesus macaques

In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.