EPR and M?ssbauer studies of nucleotide-bound nitrogenase iron protein from Azotobacter vinelandii

We have recently shown (Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Münck, E. (1985) J. Biol. Chem. 260, 11160-11173) that the [4Fe-4S]1+ cluster of the native Fe protein can exist in two forms characterized by different cluster spin: an S = 1/2 state exhibiting a g = 1.94 type EPR signal and an S = 3/2 state yielding signals at g approximately 5.8 and 5.1. We have now extended our study of the Fe protein to include the MgATP- and MgADP-bound forms. The [4Fe-4S]1+ cluster of the nucleotide-bound Fe protein exists in a similar S = 1/2, S = 3/2 spin mixture. The S = 3/2 cluster exhibits a broad EPR signal at g approximately 4.8. In spectra of the MgATP-bound protein, we have also observed a g = 4.3 signal from an S = 5/2 state (D = 1 - 3 cm-1, E/D approximately 0.32). Various experiments strongly suggest that this signal does not originate from adventitiously bound Fe3+. The g = 4.3 signal may arise from approximately 2% of the [4Fe-4S]1+ clusters when MgATP is protein-bound. We have also discovered substoichiometric amounts of what appears to be ADP in some nominally nucleotide-free Fe protein samples. MgATP binds to Fe protein in the presence of perturbing solvents, resulting in EPR spectra similar to those of MgATP-bound samples in aqueous solutions; MgADP binding, on the other hand, results in signals more typical of the solvent state. Spectra of samples frozen during turnover of the nitrogenase system exhibit a mixture of spin states. Characterization of the Fe protein EPR signature described here should aid future mechanistic and nucleotide-binding studies.     

Yersinia enterocolitica and related species isolated from wildlife in New York State.

Fecal specimens for Yersinia screening were obtained from a variety of wild mammals, birds, reptiles, fish, and invertebrates throughout New York State. One specimen from each of 1,426 animals was examined. A total of 148 isolates of Yersinia enterocolitica and related species were obtained from 133 (9.3%) of the animals. Y. enterocolitica was isolated from 100 (7%) of the animals tested, including 81 (10%) of 812 mammals and 19 (3.3%) of 573 birds. Y. intermedia, Y. frederiksenii, and Y. kristensenii were isolated from 39 (2.7%), 5 (0.35%), and 4 (0.28%) animals, respectively. The 81 Y. enterocolitica isolates from mammals belonged to 15 serogroups and included three pathogens: two isolates of typical serogroup 0:8, the "American strain," one from a gray fox (Urocyon cinereoargenteus) and one from a porcupine (Erethizon dorsatum); and one isolate of serogroup 0:3, bacteriophage type IXb, the "Canadian strain," from a gray fox. The most prevalent serogroups recovered from mammals were 0:6,31 (16 isolates) and 0:5,27 (6 isolates). The 19 isolates of Y. enterocolitica from birds belonged to nine serogroups and included one serogroup 0:6,31 isolate from a common grackle (Quiscalus quiscula) and two serogroup 0:5,27 isolates from great horned owls (Bubo virginianus).     

The effect of anti-hypertensive drug treatment on brown adipocyte diameter and locule distribution in rats

To determine the effects of sympathoplegic anti-hypertensive drug treatment on brown adipose tissue morphology, groups of adult male Wistar-Kyoto (WKY) and spontaneous hypertensive (SHR) rats were orally administered a solution containing 2.3 microM reserpine, 0.5 M hydralazine and 1.68 mM hydrochlorothiazide ad libitum or tap water and brown adipocyte diameter and extent of fat loculization were determined 3 weeks later. Pulse rates of rats were significantly greater in SHR than WKY and were unaffected by treatment, while drug treatment resulted in significant decreases in mean arterial pressure of both groups at the end of the study. Mean adipocyte diameters were smaller in untreated SHR than WKY and drug treatment of both groups was associated with increases in adipocyte diameter and cytological change from physiologically more active to less active cells. These drug-induced alterations in BAT morphology area consistent with decreased sympathetic activity and suggest that thermogenic capacity of brown adipose tissue may be pharmacologically modified, thereby altering an animal's capacity for energy expenditure.     

Peroxynitrite and NO donors form colored nitrite adducts with sinapinic acid: potential applications

Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid, SA) reacted with peroxynitrous acid at neutral pH with a second-order rate constant of 812 M(-1)s(-1), to yield a red product (lambda(max), 532 nm). The identical colored product could be formed with acidified decomposed peroxynitrous acid solutions or nitrite at slower rates (0.1M HCl, 8.32 M(-1)s(-1); 10% acetic acid, 0.0004 M(-1)s(-1)). The red compound is thought to be O-nitrososinapinic acid (3,5-dimethoxy-4-nitrosooxycinnamic acid) which can be formed by reaction with either peroxynitrous acid or nitrous acid. The extinction coefficient of O-nitrososinapinic acid (ONSA) was estimated to be 8419 M(-1)cm(-1) at 510 nm in 10% acetic acid and 90% acetonitrile. ONSA was also formed via NO(+) transfer from S-nitrosoglutathione (GSNO). ONSA in turn can S-nitrosate low molecular weight thiols and protein thiols. SA was also shown to act as a peroxynitrite sink as it effectively prevented the oxidation of dihydrorhodamine under physiological conditions. The fact that O-nitrososinapinic acid is stable and can be used to S-nitrosate thiol containing amino acids, peptides, and proteins makes it a potentially useful reagent in the study of S-nitrosothiol biochemistry and physiology. In addition, the relatively high extinction coefficient of O-nitrososinapinic acid means that it could be utilized as an analyte for the spectroscopic detection of peroxynitrite or NO(+)-donors in the submicromolar range.