Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein

The yeast Rev1 protein (Rev1p) is a member of the Y family of DNA polymerases that specifically catalyzes the incorporation of C opposite template G and several types of DNA damage. The X-ray crystal structure of the Rev1p-DNA-dCTP ternary complex showed that Rev1p utilizes an unusual mechanism of nucleotide incorporation whereby the template residue is displaced from the DNA double helix and the side chain of Arg-324 forms hydrogen bonds with the incoming dCTP. To better understand the impact of this protein-template-directed mechanism on the thermodynamics and kinetics of nucleotide incorporation, we have carried out pre-steady-state kinetic studies with Rev1p. Interestingly, we found that Rev1p's specificity for incorporating C is achieved solely at the initial nucleotide-binding step, not at the subsequent nucleotide-incorporation step. In this respect, Rev1p differs from all previously investigated DNA polymerases. We also found that the base occupying the template position in the DNA impacts nucleotide incorporation more at the nucleotide-binding step than at the nucleotide-incorporation step. These studies provide the first detailed, quantitative information regarding the mechanistic impact of protein-template-directed nucleotide incorporation by Rev1p. Moreover, on the basis of these findings and on structures of the unrelated Escherichia coli MutM DNA glycosylase, we suggest the possible structures for the ternary complexes of Rev1p with the other incoming dNTPs.     

Quantitative analysis of the yeast proteome by incorporation of isotopically labeled leucine

Quantitative comparison of protein expression levels in 2D gels is complicated by the variables associated with protein separation and mass spectrometric responses. Metabolic labeling allows cells from different experiments to be mixed prior to analysis. This approach has been reported for prokaryotic cells. Here, we demonstrate that metabolic labeling can also be successfully applied to the eukaryote Saccharormyces cerevisiae. Yeast leucine auxotrophs grown on synthetic complete media containing natural abundance Leu or D10-Leu were mixed prior to 2D gel separation and MALDI analysis of the digested proteins. D10-Leu labeling provided an effective internal calibrant for peptide MS analysis, and the number of Leu residues yielded an additional parameter for peptide identification at low mass resolution (1000). Metabolic incorporation of D10-Leu into yeast proteins was found to be quantitative since the intensities of the peptide peaks corresponded to those expected on the basis of the percent label in the media. Thus, D10-Leu labeling should provide reliable data for comparing proteomes both quantitatively and qualitatively from wild-type and nonessential-gene-null-mutant strains of S. cerevisiae. Given the central role played by yeast in our understanding of eukaryotic gene and protein expression, it is anticipated that the quantitative expressional proteomic method outlined here will have widespread applications.     

Uptake and incorporation of glucose and mannose by whole cells of Bacteroides thetaiotaomicron.

Glucose uptake by whole-cell suspensions of the obligate anaerobe Bacteroides thetaiotaomicron was two- to fourfold higher under aerobic conditions than during incubation under atmospheres of N(2) or H(2) gas. The O(2)-stimulated uptake activity was lost rapidly (>70% in 5 h) when cell suspensions were incubated aerobically, but this loss was prevented by the addition of crude catalase. Catalase had no apparent effect on cell viability during these incubations. Glucose uptake activity was strongly inhibited by a 10-fold excess of mannose or galactose but not by methyl-alpha-d-glucoside, fructose, or lactose. Both glucose and mannose were rapidly incorporated into polyglucose after uptake. The O(2)-stimulated glucose uptake was not inhibited by cyanide, azide, 2,4-dinitrophenol, or 2-N-heptyl-4-hydroxyquinoline-N-oxide. However, p-chloromercuribenzoate, menadione, and sodium fluoride inhibited uptake by 88, 67, and 55%, respectively. All attempts to detect phosphoenolpyruvate-phosphotransferase activity for glucose, methyl-alpha-d-glucoside, and 2-deoxyglucose were negative. The bacteria contained hexokinase activity and a complete glycolytic Embden-Meyerhof pathway.     

Modulation of adenine nucleoside excretion and incorporation in adenosine deaminase deficient human lymphoma cells

The availability of a human lymphoma cell line deficient in adenosine deaminase, adenosine kinase and methylthioadenosine phosphorylase enabled us to compare the effects of nucleoside transport inhibitors on the excretion of endogenously generated adenosine, deoxyadenosine and 5'-methylthioadenosine. The nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole blocked the efflux of adenosine, but not deoxyadenosine or 5'-methylthioadenosine. The inhibitors also prevented the uptake of exogenous adenosine, but not deoxyadenosine or 5'-methylthioadenosine, by human lymphoblasts. The results show (i) that the transport inhibitors modify adenine nucleoside efflux and influx similarly, and (ii) that the effects of the compounds on the excretion and uptake of these three physiologically important adenine nucleosides are distinctly different.     

Tracer kinetic analysis of signal time series from dynamic contrast-enhanced MR imaging.

Rapid magnetic resonance imaging (MRI) makes it possible to detect the fast kinetics of tissue response after intravenous administration of a paramagnetic contrast medium (CM), reflecting the status of tissue microcirculation. In this paper, the basic physical and tracer kinetic principles of dynamic relaxivity and susceptibility contrast-enhanced MRI are reviewed. Quantitative analysis of data acquired is broken up into an MR-specific part, in which the signal variation observed is related to the CM concentration in the tissue, and an MR-independent part, in which the computed concentration time series are analyzed by tracer kinetic modeling to estimate well-defined physiological tissue parameters. The clinical application of dynamic MRI techniques is demonstrated by two representative studies.     

Purification and steady-state kinetic analysis of yeast thiosulfate reductase.

Thiosulfate reductase has been purified approximately 70-fold from an extract of bakers' yeast. An enzyme with a molecular weight of 17,000, a Stokes radius of 19 Å, and a pI of 5.1 was obtained. Initial velocity and inhibition studies indicate that the substrates add in a random fashion. Further evidence suggests that the rapid-equilibrium assumption is not totally applicable. The enzyme has two distinct but closely situated substrate binding sites—one for compounds with an RSO₃^? structure and one for the sulfhydryl substrate.     

Tracer studies of nitrogen assimilation in yeast

By using N(15) as a tracer the assimilation of ammonia by the yeast, Torulopsis utilis, has been studied. It has been shown that: 1. There was no measurable incorporation of N in the protein or polynucleotide purine of carbon-starved yeast. 2. When ammonia is added to nitrogen-starved yeast there is a long lag period before division begins during which the yeast rapidly synthesizes protein, this process being accompanied by a turnover of polynucleotide purine. There was no significant dilution of the N(15)H(4) (+) of the medium by ordinary NH(4) (+). 3. When yeast containing N(15) is allowed to divide and grow in ordinary ammonia, the total amount of N(15) in the yeast remains constant. The dicarboxylic amino acids are most diluted, while arginine and nucleic acid guanine are not diluted at all.     

Stopped-flow kinetic analysis of the reaction catalyzed by the full-length yeast cystathionine beta-synthase

Cystathionine beta-synthase found in yeast catalyzes a pyridoxal phosphate-dependent condensation of homocysteine and serine to form cystathionine. Unlike the homologous mammalian enzymes, yeast cystathionine beta-synthase lacks a second cofactor, heme, which facilitates detailed kinetic studies of the enzyme because the different pyridoxal phosphate-bound intermediates can be followed by their characteristic absorption spectra. We conducted a rapid reaction kinetic analysis of the full-length yeast enzyme in the forward and reverse directions. In the forward direction, we observed formation of the external aldimine of serine (14 mm(-1) s(-1)) and the aminoacrylate intermediate (15 s(-1)). Homocysteine binds to the aminoacrylate with a bimolecular rate constant of 35 mm(-1) s(-1) and rapidly converts to cystathionine (180 s(-1)), leading to the accumulation of a 420 nm absorbing species, which has been assigned as the external aldimine of cystathionine. Release of cystathionine is slow (k = 2.3 s(-1)), which is similar to k(cat) (1.7 s(-1)) at 15 degrees C, consistent with this being a rate-determining step. In the reverse direction, cystathionine binds to the enzyme with a bimolecular rate constant of 1.5 mm(-1) s(-1) and is rapidly converted to the aminoacrylate without accumulation of the external aldimine. The kinetic behavior of the full-length enzyme shows notable differences from that reported for a truncated form of the enzyme lacking the C-terminal third of the protein (Jhee, K. H., Niks, D., McPhie, P., Dunn, M. F., and Miles, E. W. (2001) Biochemistry 40, 10873-10880).     

Effect of glucose starvation on the nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase of yeast.

Yeast cells growing on mineral medium plus ammonia and glucose contained high levels of nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase activity, as measured in crude extracts. After suspension of cells in fresh medium lacking glucose, there was a loss of the glutamate dehydrogenase activity. Loss of activity was inhibited by 2,4-dinitrophenol, sodium azide, iodoacetic acid, and cycloheximide. The enzyme activity was restored when glucose was added back to the medium, and this recovery was fully prevented in the presence of cycloheximide.     

Nonselective enrichment for yeast adenine mutants by flow cytometry

The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.     

Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K+ from the cells. Hyperpolarization shows a peak at about 3 min and a net K+ efflux at 4-5 min. An additional transient hyperpolarization and net K+ efflux are found after 60-80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K+ efflux. At low pH, neither transient hyperpolarization nor a transient K+ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K+ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K+ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K+, whereas the same amount of Na+ does not reduce the hyperpolarization.     

Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K⁺ from the cells. Hyperpolarization shows a peak at about 3 min and a net K⁺ efflux at 4–5 min. An additional transient hyperpolarization and net K⁺ efflux are found after 60–80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K⁺ efflux. At low pH, neither transient hyperpolarization nor a transient K⁺ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K⁺ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K⁺ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K⁺, whereas the same amount of Na⁺ does not reduce the hyperpolarization     

Cell-cycle analysis of fission yeast cells by flow cytometry

The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.     

Phloretin inhibition of glucose transport by the tapeworm Hymenolepis diminuta: a kinetic analysis

Phloretin non-competitively inhibits glucose and methionine absorption by Hymenolepis diminuta (Ki = 0.24 and 1.48 mM, respectively). Inhibition of glucose transport by phloretin is reversible. Phloretin and phlorizin binding sites on the surface of H. diminuta are distinct. Phloretin does not inhibit Na+ transport independent of glucose transport in H. diminuta. The data suggest that phloretin inhibits absorptive functions across the surface of H. diminuta via non-specific binding sites associated with the lipid portion of the worm outer membrane.