Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Calcium Reserves in Breeding Lesser Snow Geese (Chen caerulescens caerulescens)

Calcium and fat reserves of the femur medullary bone were examined in sexually mature lesser snow geese (Chen caerulescens caerulescens) collected during the 1974–1975 season. In females, femur calcium and fat levels increased by 80 and 30%, respectively, during the spring migration, much of the increase taking place while the birds staged in southwestern Manitoba and North Dakota prior to their departure for the breeding area. In males. femur calcium levels showed no seasonal change but femur fat increased in a manner similar to that found in the females, although the increase was not as great (17%). In the females, femur fat content fell by 40% during egg-laying whereas in males a decrease in femur lipid was not evident until incubation was well underway. Femur calcium levels in females declined during egg production and early incubation, showing a 56% decrease over spring migratory levels, indicating that dietary calcium intake was limited during the nesting period. However, the low femur calcium levels in birds collected during the spring were not significantly different from those of wintering birds, suggesting that no calcium deficiencies were apparent. Plasma calcium levels in males remained relatively constant throughout the year, although there was some elevation in May. Plasma calcium levels in the females increased almost threefold during egg laying and returned to pre-laying levels during incubation. Medullary bone was evident only in reproducing females and appeared during spring migration, concomitant with increased femur weight, fat and calcium content. Medullary bone degradation commenced during the first week of incubation and no medullary bone was in evidence by molt. Calcium reserves of medullary bone accounted for only 17.2% of the calcium required for eggshell production, suggesting that. at least during the laying period, the female must depend on some exogenous source, perhaps from grit or brackish water.     

Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.