Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage

Recently, Knappe and co-workers [Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332] have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.     

A Farmer-based approach to conserving crop germplasm

In situ conservation of crop genetic resources from centers of agricultural diversity is considered. This strategy has been rejected for several reasons, but other factors make it an important potential contributor to the overall conservation effort. Case studies of potato agriculture in Peru, maize agriculture in Mexico, and rice agriculture in Thailand indicate that farmers frequently engage in de facto conservation of landraces. Five principles should guide planning of in situ conservation: complementarity with off-site conservation, minimal institutional development, continuity with existing programs, meeting the development goals of increasing income and food, and accepting germplasm as an international public good. Four means to implement on-site conservation are presented: the institutional framework; the information base; the policy framework; and the role of grassroots organizations.     

Metabolism of carotenoid pigments in birds

Carotenoid pigments are an important component in the plumage of birds. The metabolic precursors are dietary in origin but many species have the capacity to chemically modify and selectively deposit the pigments. The ensuing plumage patterns are important in communication and identification. The bright yellows, oranges, and reds are due mostly to xanthophylls; keto and hydroxy carotenes. Some are deposited unmodified (e.g., lutein) whereas others are modified chemically (canthaxanthin, astaxanthin). Early workers concentrated on demonstrating that feather carotenoids were derived from the diet and deposited selectively. Progress in defining and solving biological problems depended on advances in chemical and analytical techniques. Subsequent investigation showed that various plumage colormorphs, seasonal plumage changes or colors in common mutant, were due to relatively simple chemical changes in carotenoids but had profound biological consequences. Equally important was the realization that many of these processes were under genetic control. Validation came from feeding studies of flamingos and finches. Recent studies have employed the plumage carotenoids to test hypotheses of genetic divergence, to relate plumage color to environmental process, and to demonstrate the influence of synthetic changes on color. Understanding the processes has advanced with the introduction of high-resolution separation techniques and the ability to determine both conformation and absolute configuration. The next steps will be in the direction of understanding the enzymatic modification, transport, and tissue selectivity of feather carotenoids.     

Keepers of maize in Chiapas,Mexico

This study concerns the knowledge and selection of biological diversity of maize (Zea mays) within its center of domestication in Mesoamerica. Maize farmers in central Chiapas of Southern México keep local varieties (landraces) belonging to six races and four race mixtures. Fifteen local varieties are recognized. In spite of widespread adoption of a modern, high yielding variety, maize farmers continue to select local varieties for specific soils and because of agronomic and use criteria. Farmers maintain maize varieties primarily through seed selection. Spatial and temporal separation do not seem sufficient to maintain varieties. The management of improved varieties can lead from a uniform population to a heterogeneous one as hybridization with local maize populations occurs.     

Characterization and Quantification of Intrinsic Ice Nucleators in Winter Rye (Secale cereale) Leaves

Extracellular ice formation in frost-tolerant organisms is often initiated at specific sites by ice nucleators. In this study, we examined ice nucleation activity (INA) in the frost-tolerant plant winter rye (Secale cereale). Plants were grown at 20[deg]C, at 5[deg]C with a long day, and at 5[deg]C with a short day (5[deg]C-SD). The threshold temperature for INA was -5 to -12[deg]C in winter rye leaves from all three growth treatments. Epiphytic ice nucleation-active bacteria could not account for INA observed in the leaves. Therefore, the INA must have been produced endogenously. Intrinsic rye ice nucleators were quantified and characterized using single mesophyll cell suspensions obtained by pectolytic degradation of the leaves. The most active ice nucleators in mesophyll cell suspensions exhibited a threshold ice nucleation temperature of -7[deg]C and occurred infrequently at the rate of one nucleator per 105 cells. Rye cells were treated with chemicals and enzymes to characterize the ice nucleators, which proved to be complexes of proteins, carbohydrates, and phospholipids, in which both disulfide bonds and free sulfhydryl groups were important for activity. Carbohydrates and phospholipids were important components of ice nucleators derived from 20[deg]C leaves, whereas the protein component was more important in 5[deg]C-SD leaves. This difference in composition or structure of the ice nucleators, combined with a tendency for more frequent INA, suggests that more ice nucleators are produced in 5[deg]C-SD leaves. These additional ice nucleators may be a component of the mechanism for freezing tolerance observed in winter rye.     

血红细胞超氧化物歧化酶(SOD)制备工艺的研究初报

本文报告了本实验室设计的由血红细胞自溶液60℃热变性, 乙醇——氯仿法除血红蛋白,旋转蒸发法减压浓缩抽去氯仿、乙醇,硫酸铵分级盐析法沉降SOD,Sepbadex G-75层析提纯SOD等步骤构成的一条成本低、设计合理、简便实用的分离纯化SOD的工艺路线。     

Effects of acute and chronic restraint and estrus cycle on pituitary-adrenal function in the rat

Female Long-Evans hooded rats with 5-day estrus cycles were subjected to 4 hr of continuous restraint for either 1 or 20 days. On the last day of the stress regimen, plasma and adrenal corticosterone concentrations were determined and classified according to the stage of the estrous cycle. The results indicated that acute stress produced greater plasma corticosterone concentrations than controls only during estrus, whereas in response to chronic stress significant stress-induced increments were observed during estrus and proestrus. The results suggest that the estrous cycle influences the magnitude of the stress-induced increments for both acute and chronic stress. In addition, the pituitary-adrenal system did not show habituation to repeated administration of this stress, but sensitization was observed during proestrus.     

Affinity of Ionic Species of Nucleoside Transport Protein Inhibitors

Abstract

A class of very potent nucleoside transport inhibitors is present in two molecular forms around physiological pH. We investigated whether the monoprotonated or the unionized species of these molecules binds to this camer protein with higher affinity.