A mutation altering the kinetic responses of the yeast mitochondrial F1-ATPase

Nucleotide-binding proteins, including the mitochondrial F1-ATPase, the ras proteins, and the G-proteins, contain a homologous glycine-rich sequence that is thought to constitute part of the active site. This study reports the effects of a single amino acid replacement of Thr197 to Ser197, which is located at the hinge region of this putative loop, in the yeast Saccharomyces cerevisiae F1-ATPase. This replacement resulted in a 3-fold increase in the specific activity of the enzyme, eliminated the stimulatory effects of oxyanions, and modulated the effects of the inhibitor NaN3 while having little effect on the uni-site ATPase. These results indicate a role of the glycine-rich loop in many of the kinetic responses of the F1-ATPase.     

Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reversed-phase high-performance liquid chromatography, and contrasting results to immunoassays

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible ODS columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in order to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination of each sample with two newly developed separation methods: (a) pre-separation with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ultrafilter followed by the last two steps in (a). For detection UV was preferred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectrum of results ranging from good to untenable without any warning whatever about functionality. The measurement of official controls, with reference values derived via gas chromatography-isotope dilution mass spectrometry, also demonstrated the superiority of the double HPLC method.     

Comparison of the uptake of vitamin B12 by Spirometra mansonoides and Hymenolepis diminuta and the functional groups of B12 analogs affecting uptake.

Uptake of 57Co-vitamin B12 (CN-Cbl) by spargana (larvae) of the pseudophyllidean tapeworm, Spirometra mansonoides, was affected by temperature, was saturable with respect to concentration of CN-Cbl in the medium, and was inhibited in the presence of several structural analogs of CN-Cbl. In uptake studies with various analogs it was found that chemical modifications which altered the benzimidazole moiety greatly reduced the ability of the worm to take up these analogs. Modifications in which the amide groups of the propionamide side chains were removed, resulting in carboxylic acid derivatives, showed greatly reduced transport properties. The C-13 epimer in which the e-proprionamide side chain is no longer on the benzimidazole side (lower) of the molecule but is inverted to a position on the upper side was freely taken up. The pharmacological implications of this last observation are discussed. Adult Hymenolepis diminuta did not take up CN-Cbl in vitro, which correlated with the finding that no CN-Cbl was detected in the worm by Ochromonas malhamensis assay.     

The relationship between growth and reversion in the Ames Salmonella plate incorporation assay

Growth curves of the 5 commonly used Ames Salmonella tester strains have been measured turbidimetrically in semi-solid agar. Lag times, doubling times and maximum cell densities have been calculated for each of the 5 strains. The time dependence of reversion has been studied in the standard plate incorporation assay using 1-h pulsed doses of (a) bromoethane, a volatile chemical mutagen, and (b) 1-h exposures to visible light. Essentially no reversion takes place during the first 4 h after plating. Reversion is detectable between hours 4 and 16. The cumulative or integrated revertants versus time curve has the characteristics of a growth curve. Conversely the derivatives of the growth curves resemble the curves obtained in the pulsed mutagenicity studies. Thus, the reversion rate in any given 1 h interval is proportional to the growth rate during that same interval. These results suggest that mutagenic chemicals must be present during the bacterial growth cycle (about 4-16 h after plating) in order to revert the tester strains. Short-lived chemical mutagens, then, should produce enhanced results if plated 6-8 h after the bacteria. We have confirmed this for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 9-aminoacridine and 2-aminoanthracene (with S9).     

Phosphorus-31 nuclear magnetic resonance of insect hemolymph sera

Sera from larval and pupal stages of the tobacco hornworn, Manduca sexta, have been investigated using phosphorus-31 pulsed Fourier transform nuclear magnetic resonance. Spectra of larval and pupal sera containing 5 mm EDTA were characterized by four major peaks and one or more minor resonances. A phosphorus-31 spectrum of dialyzed larval serum showed several weak signals which indicated the presence of some higher-molecular-weight phosphorylated compounds as well. None of those signals, however, corresponded to any of the ones seen with undialyzed sera. Three of the four prominent peaks and one minor peak in the whole larval serum had the same chemical shifts as those in the pupal samples. The pupal sera, in addition, displayed an extra peak well upfield from those of the larval stage. All of the low-molecular-weight resonances detectable in the hemolymphs have been identified and included four compounds not previously reported; trehalose-6-phosphate, phosphoarginine, phosphatidylcholine, and phosphatidylethanol-amine. The phosphometabolites found at millimolar or higher concentrations in larval hemolymph were α-glycerolphosphate, phosphorylcholine, phosphorylethanolamine, inorganic phosphate, trehalose-6-phosphate, phosphatidylcholine, and phosphatidylethanolamine. All of the above compounds were found in pupal sera as well except for the addition of phosphoarginine and the deletion of phosphorylethanolamine. The levels of the phosphometabolites in common between the two stages of development, however, were quite different as were their stabilities after extraction. While the intensities of the larval phosphates remained virtually constant in the presence of EDTA at pH 7.8, those of the pupal sera changed rapidly. This was especially true for arginine phosphate which disappeared quickly.     

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.     

Purification and properties of human placental acid lipase

Two peaks of lysosomal acid lipase activity were purified from normal human placenta. Acid lipase I, with an estimated molecular weight of 102 500, was purified 1016-fold while acid lipase II, with an estimated molecular weight of 30 600, was purified 3031-fold. The final yields of enzyme activity for acid lipase I and II were 0.9% and 2.2% respectively. The purity of the final preparations was documented by demonstration of a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both preparations of the purified enzyme demonstrated activity towards triolein, cholesteryl oleate and the artificial substrate 4-methylumbelliferyl oleate. Examination of Km values, thermal stability, pH optima, and electrophoretic mobility revealed similar properties for the two enzyme peaks. The response of the two enzyme preparations to inhibitors was similar with both being significantly inhibited by 0.2 M NaCl, 0.2 M KCl, 5 mM HgCl2 and 5 mM p-chloromercuribenzoate. The activity of the two preparations as assayed with either triolein or cholesterol oleate was not significantly affected by the addition of bovine serum albumin. In contrast, the 4-methylumbelliferyl oleate activity of both preparations was significantly inhibitred by albumin. These findings support the hypothesis that the same enzyme or enzymes are responsible for the intralysosomal hydrolysis of triacylglycerols and cholesterol esters in human tissues.     

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.