A two-faced molecule offers NO explanation: the proximal binding of nitric oxide to haem.

Cytochrome c ' (cyt c ') is found in the periplasmic space of denitrifying bacteria where it is thought to mediate the transfer of NO between the nitrogen-cycle enzymes dissimilatory nitrite reductase and nitric oxide reductase. It contains a 5-coordinate (5c) His-ligated haem that shares spectroscopic and ligand-binding properties with the haem group in the sensory domain of soluble guanylate cyclase (sGC). The latter is an extremely important enzyme involved in the control of vasodilation and blood clotting. Curiously, the enzyme is activated up to 200-fold by the binding of NO to the haem, whereas the binding of CO gives rise to only a mild stimulation of activity. Through X-ray crystallography we have studied NO and CO binding to cyt c '. CO binds to the distal face to give a 6-coordinate (6c) adduct. By contrast, NO binding gives rise to a 5c adduct through the displacement of the proximal His, to give a novel and unexpected proximal binding mode for NO. These results are also supported by a range of spectroscopies. In the absence of a crystal structure for sGC we propose that cyt c ' provides a structural model for the haem domain of this enzyme and thereby helps to explain the differential effects of NO and CO on its activity.

GnRH and gonadotropin release is decreased in chronic nitric oxide deficiency.

Nitric oxide synthetase (NOS), the conversion enzyme for nitric oxide (NO) is localized in the anterior pituitary of female rats, particularly in gonadotrophs and folliculo-stellate cells, suggesting that NO regulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary. The focus of this study was to determine the effect of chronic NO deficiency on the subsequent pituitary release of LH and FSH in vitro and the hypothalamic immunoexpression of GnRH in vivo. NO deficiency was induced by adding the NOS inhibitor, N-nitro-L-arginine (L-NNA, 0.6 g/L) to the drinking water of female Wistar rats. After 8 weeks, the animals were euthanized, the pituitaries were removed, and they were incubated in vitro. Pituitaries were perfused for 4 hr in the presence of pulsatile gonadotropin release hormone (GnRH, 500 ng/pulse) every 30 min. S-Nitroso-L-acetyl penicillamine (SNAP, an NO donor, 0.1 mM) or L-nitro-argine methyl ester (L-NAME, a NOS inhibitor, 0.1 mM) was added to the media and perfusate samples were collected at 10-min intervals. LH and FSH levels in the perfusate were measured by double antibody radioimmunoassays. Pituitaries from the NO-deficient rats had a significantly smaller GnRH-stimulated release of LH and FSH compared with proestrous control rats. The addition of S-NAP to the perfusate resulted in decreased LH and FSH secretion in the control group, but increased LH secretion in the NO-deficient group. The addition of L-NAME to the perfusate suppressed LH secretion from control pituitaries, but not in pituitaries from NO-deficient animals. Immunohistochemistry of brain slices demonstrated that NO-deficient rats had a large qualitative decrease of GnRH in the median eminence compared with their controls. This decrease was particularly evident in the external capillary plexus of the median eminence. We concluded that chronic NO deficiency is associated with a decreased GnRH in neurosecretory terminals in the external capillary layer of the median eminence, accompanied by a decrease in LH and FSH release from the pituitaries.

Recovery from carotid artery catheterization performed under various anesthetics in male, Sprague-Dawley rats.

This study was designed to determine the time to recovery from carotid artery catheterization using multiple criteria and to compare recovery times between three common anesthetics. Male Sprague-Dawley rats, chronically instrumented with radio-telemetry transmitters, were anesthetized with sodium pentobarbital, halothane or a mixture of ketamine, xylazine and acepromazine before an indwelling catheter was placed in the carotid artery. The procedure was completed in less than 15 min. Changes in body weight, food and water consumption, blood pressure, heart rate and activity were used to determine recovery. As judged by recovery of body weight, animals anesthetized with each of the anesthetics recovered by the 4th day after catheterization. Food and water consumption normalized by 1-2 days after surgery. Heart rates and blood pressures during the light phase of the photoperiod were significantly increased for 2 days by all anesthetics. During the dark phase of the photoperiod, heart rates and blood pressures were not significantly affected by pentobarbital or halothane anesthesia, but were significantly decreased and increased respectively on the night immediately following surgery in the ketamine / xylazine / acepromazine-anesthetized rats. Delayed elevations of heart rate were observed in pentobarbital and halothane anesthetized rats on days and/or nights 5 and 6 post surgery. Animal activity patterns during the light phase of the photoperiod were not affected by pentobarbital or halothane, but were increased by ketamine 2 days after surgery. During the dark phase, halothane transiently reduced activity whereas ketamine-anesthetized rats showed reduced activity for 4 nights post surgery. These studies show that recovery depends on the criteria selected and the anesthetic used, but, in general, 2-4 days were required for recovery from this relatively simple procedure.

Differences in plasma and nipple aspirate carotenoid by lactation status.

BACKGROUND: Dietary antioxidants, such as provitamin A carotenoid, have a protective effect against breast cancer. The transport of carotenoid from the blood into the breast microenvironment may be enhanced by lactation. OBJECTIVE: To examine the association between plasma and nipple aspirate carotenoid levels by lactation and post-wean status. METHODS: The sample consisted of 43 women, ages 18-45, who were at least 12 months postpartum. Women who had breastfed their last infant were at least 3 months post-wean. Women collected breast fluid every other day for 17 days and had a venipuncture for total nipple aspirate and plasma carotenoid, and completed a written health assessment. RESULTS: The association between plasma and nipple aspirate carotenoid levels was significant for breastfeeding women (r =.39, p=.03), but not for non-breastfeeding women (r =.31, p =.27). However, while the association between plasma and nipple aspirate carotenoid levels was significant for women at or less than 9 months post-wean (r =.65, p = .01), the effect for women after 9 months post-wean (r = .21, p =.45) was not significant. CONCLUSION: Lactation may be protective by enhancing the delivery of chemopreventive substances available in the blood to the cell level of the breast, even after breast involution has occurred post lactation.

Two crystal structures of the cytoplasmic molybdate-binding protein ModG suggest a novel cooperative binding mechanism and provide insights into ligand-binding specificity.

The X-ray structures of the cytoplasmic molybdate-binding protein ModG from Azotobacter vinelandii in two different crystal forms have been determined. For such a small protein it is remarkably complex. Each 14.3 kDa subunit contains two small beta-barrel domains, which display an OB-fold motif, also seen in the related structure of ModE, a molybdenum-dependent transcriptional regulator, and very recently in the Mop protein that, like ModG, has been implicated in molybdenum homeostasis within the cell. In contrast to earlier speculation, the functional unit of ModG is actually not a dimer (as in ModE), but a trimer capable of binding a total of eight molybdate molecules that are distributed between two disparate types of site. All the binding sites are located at subunit interfaces, with one type lying on a crystallographic 3-fold axis, whilst the other lies between pairs of subunits. The two types of site are linked by short hydrogen bond networks that may suggest a cooperative binding mechanism. A superposition of two subunits of the ModG trimer on the apo-ModE dimer allows the probable locations of the molybdate-binding sites of the latter to be assigned. Through structural comparisons with other oxyanion-binding proteins, including Mop and ModE, it is possible to speculate about ligand-binding affinities, selectivity and evolution.

The effects of routine cage-changing on cardiovascular and behavioral parameters in male Sprague-Dawley rats.

The objective of this study was to determine whether the blood pressure and heart rate of adult male Sprague-Dawley rats are affected by the routine animal husbandry procedure of moving animals to clean cages. Cardiovascular parameters were obtained by using radiotelemetry; behavior in the home cage also was evaluated. Each rat had a radiotelemetry transmitter implanted in the peritoneal cavity, with the attached catheter placed in the femoral artery. After a 7- to 9-day recovery period, half of the rats were moved to clean cages with fresh wood-chip bedding; the other animals were left undisturbed. Systolic, diastolic, and mean arterial blood pressures; heart rate; and cage behavior (movement, rearing, grooming) increased promptly and significantly when animals were placed in clean cages. These cardiovascular and behavioral responses lasted for 45 to 60 min. Those animals not moved to clean cages but present in the animal room when this procedure was done did not show significant increases in blood pressure, heart rate, or activity. When rats were moved to clean cages that contained new bedding plus a small quantity of the soiled bedding from their previous cage, the cardiovascular and behavioral responses were similar to those of animals exposed to completely fresh bedding. The responses of rats being moved to new cages did not diminish between the first and fourth weekly cage change. Rats whose cages were not changed for 2 weeks showed small, but significant, increases in cardiovascular and behavioral responses above the responses in animals with weekly cage changes. We conclude that ordinary animal husbandry procedures such as moving rats to a clean cage can induce transient, but significant, cardiovascular and behavioral changes. Investigators and animal care staff should recognize that such routine procedures could confound experiments conducted shortly thereafter.

Resonance Raman studies of cytochrome c' support the binding of NO and CO to opposite sides of the heme: implications for ligand discrimination in heme-based sensors.

Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidans cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO and CO but not O(2). Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sides of its heme (distal and proximal) for binding exogenous ligands to its Fe center. The present RR investigation of the Fe coordination and heme pocket environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' in solution fully supports the structures determined by X-ray crystallography and offers insights into mechanisms of ligand discrimination in heme-based sensors. Ferrous cytochrome c' reacts with CO to form a six-coordinate heme-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively high stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond. RR spectra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal heme pocket. On the other hand, RR data for the five-coordinate NO adduct suggest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme. Parallels between certain physicochemical properties of cytochrome c' and those of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO binding.

Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase.

We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13-lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null-effect of mutating pea 9/13-lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9-lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13-lipoxygenase and cucumber 13/9-lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9-lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate-binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxygenases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers.

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Intergenerational transmission: individuation and intimacy across three generations.

This study examined the transmission of intergenerational family processes across three generations, employing Williamson's construct of Personal Authority in the Family System (PAFS) as a theoretical back-drop. From a PAFS perspective, psychological health is viewed as directly related to the degree of individuation and intimacy (PAFS) experienced within the family of origin. Overall, the results provided a degree of support for the intergenerational transmission hypothesis. The strongest predictor of the transmission process was from the participant/parent relationship to the participant/spouse relationship (spousal fusion/individuation). Separate male and female analyses of the Spousal Fusion/Individuation model found a moderate effect for females and a large effect for males. A small effect was found in predicting nuclear family triangulation from parent and spouse variables, although there was no gender effect. The findings suggest that degree of individuation and its related constructs are more critical in the transmission process than is intimacy.

Structural origins of the interfacial activation in Thermomyces (Humicola) lanuginosa lipase.

The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.

Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase.

Microbial cytochromes c' contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 A resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His-Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species. In contrast, CO binds 6-coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c' are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.

Crystal structure of the molybdenum cofactor biosynthesis protein MobA from Escherichia coli at near-atomic resolution.

BACKGROUND: All mononuclear molybdoenzymes bind molybdenum in a complex with an organic cofactor termed molybdopterin (MPT). In many bacteria, including Escherichia coli, molybdopterin can be further modified by attachment of a GMP group to the terminal phosphate of molybdopterin to form molybdopterin guanine dinucleotide (MGD). This modification reaction is required for the functioning of many bacterial molybdoenzymes, including the nitrate reductases, dimethylsulfoxide (DMSO) and trimethylamine-N-oxide (TMAO) reductases, and formate dehydrogenases. The GMP attachment step is catalyzed by the cellular enzyme MobA. RESULTS: The crystal structure of the 21.6 kDa E. coli MobA has been determined by MAD phasing with selenomethionine-substituted protein and subsequently refined at 1. 35 A resolution against native data. The structure consists of a central, predominantly parallel beta sheet sandwiched between two layers of alpha helices and resembles the dinucleotide binding Rossmann fold. One face of the molecule bears a wide depression that is lined by a number of strictly conserved residues, and this feature suggests that this is where substrate binding and catalysis take place. CONCLUSIONS: Through comparisons with a number of structural homologs, we have assigned plausible functions to several of the residues that line the substrate binding pocket. The enzymatic mechanism probably proceeds via a nucleophilic attack by MPT on the GMP donor, most likely GTP, to produce MGD and pyrophosphate. By analogy with related enzymes, this process is likely to require magnesium ions.

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Prolactin regulation of tuberoinfundibular dopaminergic neurons: immunoneutralization studies.

The purpose of this study was to determine the effects of acute hypoprolactinemia on tuberoinfundibular dopamine (DA) neurons using a rabbit anti-rat prolactin antiserum (PRL-AB) to immunoneutralize circulating prolactin under basal conditions and at various times after haloperidol-induced hyperprolactinemia. The specificity of PRL-AB for prolactin was determined by examining the ability of unlabelled hormone to displace binding of 125I-labelled prolactin to PRL-AB. Tuberoinfundibular DA neuronal activity was estimated by measuring the concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence which contains terminals of these neurons. Systemic (i.v.) administration of 200 microl of PRL-AB decreased plasma prolactin concentrations below detectable levels for at least 4 h, and this was accompanied by a pronounced decrease in DOPAC concentrations in the median eminence of females, but not males. Central (i.c.v.) administration of 2 microl PRL-AB diluted up to 1:100 mimicked the inhibitory effect of systemic administration of PRL-AB on median eminence DOPAC concentrations suggesting that the tonic stimulatory effect of prolactin on the basal activity of tuberoinfundibular DA neurons in females occurs via a central site of action. In male rats, blockade of anterior pituitary DA receptors with haloperidol (1 mg/kg; s.c.) caused an prompt (by 1 h) increase in plasma prolactin concentrations which was maintained for at least 12 h. Haloperidol-induced hyperprolactinemia also caused a delayed (at 6 and 12 h) increase in median eminence DOPAC concentrations in these animals which was blocked by PRL-AB. Exposure of rats to initial priming periods of endogenous hyperprolactinemia of up to 6 h duration (followed by 6 h or more of PRL-AB-induced hypoprolactinemia) failed to alter median eminence DOPAC concentrations unless prolactin exposure was reinstated by an i.c.v. injection of prolactin. These results confirm that prolactin mediates the stimulatory effects of haloperidol on tuberoinfundibular DA neurons, and reveal that delayed induced activation of these neurons by prolactin is dependent upon a priming period of sustained hyperprolactinemia longer than 3 h for initiation and maintenance of this response.

The effects of housing enrichment on cardiovascular parameters in spontaneously hypertensive rats.

By using radiotelemetry, we measured blood pressure, heart rate, and locomotor activity in adult male spontaneously hypertensive (SHR) rats during three consecutive periods in which they received various social and non-social cage enrichments. The objective was to determine whether these enriching experiences would affect cardiovascular parameters. During the first period, the readings from four individually housed males, each with a telemetry transmitter in the abdominal cavity and connected to a femoral artery catheter, were compared to those from five similarly instrumented rats that were each housed with another rat. Systolic blood pressure and activity but not diastolic blood pressure or heart rate were higher in rats housed with another rat compared to those housed alone. During the second period, each cage of animals was enriched by including a large piece of plastic drainpipe and several golf balls. In addition, the nine animals were placed together daily for two hours at the beginning of the dark phase of the photoperiod in a large, three-tiered enclosure containing a running wheel, several lengths of plastic drainpipe, and multiple golf balls. Systolic and diastolic blood pressures but not heart rate or activity were higher in the double-housed rats than those housed alone. During the last period, the rats previously housed with another rat were switched to single housing, and those previously housed alone were placed with another rat. The daily activity and cage enrichments were otherwise continued. Removal of a cage mate increased systolic blood pressure, diastolic blood pressure, and heart rate but not activity compared parameters in animals that were changed from single to double housing. During the entire experiment, activity and all cardiovascular parameters were increased during the dark phase compared to the light phase of the daily photoperiod. However, there was no statistically significant correlation between these circadian changes and the housing conditions. In summary, providing social enrichment in the form of another rat or non-social cage enrichment combined with a daily period of group housing and physical activity increased diastolic and/or systolic blood pressure of SHR rats. In addition, the loss of continuous social enrichment increased blood pressure and heart rate even when the other enrichments were continued. These changes were not always related to increased activity in the cage.

New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein.

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.

Crystallization and preliminary X-ray studies on the molbindin ModG from Azotobacter vinelandii.

Crystals of the molbindin ModG (subunit Mr = 14359 Da), a cytoplasmic molybdate-binding protein from Azotobacter vinelandii, were grown by vapour diffusion. Both apo and tungstate-bound forms were crystallized and X-ray data were collected at 100 K. Apo-ModG crystallizes in space group P6322, with unit-cell dimensions a = b = 90.62, c = 79.46 A. Native data to a resolution of 2.5 A were collected from a single crystal, which showed a marked improvement in diffraction quality after annealing. Data from a single-site gold derivative were also collected at 2.7 A resolution. Crystals of the ligand-bound form of ModG belong to space group P321, with unit-cell parameters a = b = 50.57, c = 79.29 A. X-ray data to a resolution of 2.0 A were collected.

Ovarian hormone secretory response to gonadotropins and nitric oxide following chronic nitric oxide deficiency in the rat.

Ovarian hormone secretion is regulated by gonadotropins, and it has been demonstrated that this response is modulated by nitric oxide (NO). The focus of this study was to determine the effect of chronic NO deficiency on the secretion of ovarian steroids. Female rats were given N-nitro-L-arginine (L-NNA; 0.6 g/L) in their drinking water, and vaginal smears were obtained daily. By 4 wk of treatment, all the rats were in constant estrus or proestrus. At 6-8 wk the animals were killed; the ovaries were removed and incubated in the presence of eCG (1 IU/ml) and hCG (1 IU/ml) and/or S-nitroso-L-acetyl penicillamine (an NO donor, S-NAP; 0.1 mM) for 4 h. Medium was collected at 30-min intervals, and estradiol, progesterone, and androstenedione were measured. Ovaries from proestrous rats served as controls. Ovaries from L-NNA-treated animals had a greater basal and gonadotropin-stimulated release of estradiol but not of androstenedione or progesterone in comparison to ovaries from untreated controls. S-NAP decreased the gonadotropin-stimulated estradiol, progesterone, and androstenedione in ovaries from NO-deficient rats. Steroid secretion in controls was not responsive to S-NAP. We conclude that chronic NO inhibition produces constant estrus due to increased estradiol production and that NO acts to inhibit estradiol and androstenedione production.