The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.     

Physiological responses of soybean plants grown in a nitrogen-free or energy limited environment

Soybean (Glycine max [L.] Merr.) seedlings grown in the absence of combined N and in an Ar:O₂ (79:21, volume/volume) atmosphere had greater seedling and nodule mass, threefold higher acetylene reducing activity per gram fresh weight nodules, no observable increase in nitrogenase Fe-protein, and a higher energy charge than did control plants. A sharp fall in acetylene reducing activity and energy charge accompanying stem-girdling was prevented by exogenous succinate, a result consistent with a path from the roots to the nodule other than via the phloem.     

Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases

Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta). In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity. To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses. cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated. Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit. Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit. Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively. In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II. Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity. Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.     

Predicting greater sage‐grouse habitat selection at the southern periphery of their range

Mapping suitable habitat is an important process in wildlife conservation planning. Species distribution reflects habitat selection processes occurring across multiple spatio‐temporal scales. Because habitat selection may be driven by different factors at different scales, conservation planners require information at the scale of the intervention to plan effective management actions. Previous research has described habitat selection processes shaping the distribution of greater sage‐grouse (Centrocercus urophasianus; sage‐grouse) at the range‐wide scale. Finer‐scale information for applications within jurisdictional units inside the species range is lacking, yet necessary, because state wildlife agencies are the management authority for sage‐grouse in the United States. We quantified seasonal second‐order habitat selection for sage‐grouse across the state of Utah to produce spatio‐temporal predictions of their distribution at the southern periphery of the species range. We used location data obtained from sage‐grouse marked with very‐high‐frequency radio‐transmitters and lek location data collected between 1998 and 2013 to quantify species habitat selection in relation to a suite of topographic, edaphic, climatic, and anthropogenic variables using random forest algorithms. Sage‐grouse selected for greater sagebrush (Artemisia spp.) cover, higher elevations, and gentler slopes and avoided lower precipitations and higher temperatures. The strength of responses to habitat variables varied across seasons. Anthropogenic variables previously reported as affecting their range‐wide distribution (i.e., roads, powerlines, communication towers, and agricultural development) were not ranked as top predictors at our focal scale. Other than strong selection for sagebrush cover, the responses we observed differed from what has been reported at the range‐wide scale. These differences likely reflect the unique climatic, geographic, and topographic context found in the southern peripheral area of the species distribution compared to range‐wide environmental gradients. Our results highlight the importance of considering appropriateness of scale when planning conservation actions for wide‐ranging species.     

The subunit structure of rat liver pyruvate kinase

The amino acid composition for rat liver pyruvate kinase is reported. Thin layer peptide mapping of the tryptic digests yields 44 ninhydrin-reactive peptides, which is one-quarter the total number of lysyl and arginyl residues. No amino-terminal residue has been detected using the dansyl chloride procedure. Acid urea disc gel electrophoresis of the protein subunits yields only one protein band; yet, isoelectric focusing of the subunits in urea yields two protein bands. These results suggest that pyruvate kinase (L-type isozyme) consists of four subunits of similar primary structure, but with sufficient microheterogeniety to be able to resolve two types of subunits upon isoelectric focusing.