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1.
Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance a and the apparent electromotive force E Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E Na and increased a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of a and E Na will require characterization of the partial ionic conductances on perturbation of .  相似文献   

2.
This review summarizes our experiments on the significance of the -subunit in the functional expression of Na+/K+-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na+/K+-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH2-terminal domain to the extracellular COOH-terminus: the NH2-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na+/K+-ATPase by Na+/K+- and H+/K+-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na+/K+-ATPase -subunit and from Ile89 to the COOH–terminus of the H+/K+-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na+/K+-ATPase.  相似文献   

3.
Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [35S]GTPS with a relatively high affinity (Kd10nM). The binding was saturable and specific as it was indicated by the displacement of bound [35S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [35S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [35S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [35S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - G protein hetero-trimeric GTP binding protein with , , subunits - Gn protein GTP binding protein detected on nitrocellulose blots - GTPS guanosine 5-[-thio]triphosphate - IAA 3-indoleacetic acid - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis  相似文献   

4.
    
-Crystallin is a common lens protein of most vertebrate eye lenses and the major protein component in lenses of fishes and in many mammalian species during embryonic and neonatal stages. To facilitate the structural characterization of -crystallin possessing extensive charge heterogeneity, a cDNA mixture was constructed from the poly(A)+ mRNA isolated from shark eye lenses, and amplification by polymerase chain reaction (PCR) was carried out to obtain cDNAs encoding multiple shark -crystallins. Sequencing analysis of multiple positive clones containing PCR-amplified inserts revealed the presence of a multiplicity of isoforms in the -crystallin class of this cartilaginous fish. It was of interest to find that two shark cDNA sequences coexist, one encoding -crystallin (M1) of high methionine content (15.5%) and the other encoding one (M2) of low methionine content (5.1%), each corresponding to the major teleostean and mammalian -crystallins, respectively. Comparison of protein sequences encoded by these two shark cDNAs with published sequences of -crystallins from mouse, bovine, human, frog, and carp lenses indicated that there is about 61–80% sequence homology between different species of the piscine class, whereas only 47–66% is found between mammals and shark. A phylogenetic tree constructed on the basis of sequence divergence among various -crystallin cDNAs revealed the close relatedness between shark M2-crystallin and mammalian -crystallins and that between shark M1 and teleostean -crystallins. The results pointed to the fact that ancestral precursors of -crystallins were present in the sharp lens long before the appearance of modern-day mammalian and teleostean -crystallins.  相似文献   

5.
Solute mobilities of 28 compounds in isolated cuticular membranes (CM) from Capsicum annuum L. fruit, Citrus aurantium L. and Pyrus communis L. leaves were studied using unilateral desorption from the outer surface. First-order rate constants of desorption (k*), which are directly proportional to the diffusion coefficient in the waxy outer limiting skins of cuticles were measured. When log k* was plotted vs. molar volumes of test compounds linear graphs were obtained. The y-intercepts of these graphs (k*) represent the mobility of a hypothetical molecule having zero molar volume and the slopes of the graphs () represent the size selectivity of the barrier and are related to the free volume available for diffusion. Thus, solute mobilities in cuticles are composed of two independent terms which are subtractive. If k* and are known, k* can be estimated for any solute from its molar volume (Vx) using the equation log k*=log k* –Vx. These parameters were used to analyse the effects of plant species, extraction of cuticular waxes and molecular structure of solutes on solute mobilities in plant cuticles. For aliphatic solutes, k* was a factor of 10 smaller than for cyclic compounds, while was 0.011 and 0.012, respectively. The k*-values for CM of the three species were very similar, but was higher for bitter-orange CM (0.012) than for those of pepper fruits and pear leaves (0.009). This has the consequence that differences in solute mobilities (k*) among cuticles from different plan species increase with increasing molar volumes of solutes. Our data and our analysis provide evidence that constituents of cuticular waxes are mobile, at least in the solid amorphous wax fraction, but mobility decreases rapidly with increasing molar volume. For instance, if amounts to 0.01, mobilities of wax monomers decrease by a factor of 10 for every increase in molar volume of 100 cm3 · mol–1. Thus, hexadecanoic acid is quite mobile in the amorphous wax fraction of Citrus (k*=1.5×10–6·s–1), but for dotriacontane having twice the molar volume, k* was only 2.5×10–9·s–1, which is almost three orders of magnitude smaller. Wax esters have even higher molar volumes and their mobilities will be even smaller (about 4×10–12·s–1 for a C48-ester). Since low chain mobilities are a prerequisite for low mobilities and permeabilities, the selective advantage of high-molecular-weight wax monomers in plant cuticular waxes becomes obvious. Extracting cuticular waxes from pear leaf CM increased solute mobilities by a factor of 182, but it had no effect on size selectivity. We interpret this result as evidence to the effect that cuticular waxes reduce mobility by increasing tortuosity of the diffusion path, rather than by decreasing the mean free path of diffusional jumps and jump frequencies of diffusants.Abbreviations CM cuticular membrane(s) - 2,4-D 2,4-dichloro-phenoxyacetic acid - LAB lactic acid buffer - MX polymer matrix membranes - UDOS unilateral desorption from the outer surface  相似文献   

6.
Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na+ (K m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K m for Na+ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.  相似文献   

7.
By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12+ cells) infiltrate the tumor mass. ER-MP12+ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12+ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8+, but not the CD4+, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8+ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois  相似文献   

8.
    
An 1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linkedN-acetylglucosaminyl residue of an N-glycan, forming an 1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3 GlcNAc2. Fucose in 1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose.N-acetyllactosamine, lacto-N-biose andN-acetylchito-oligosaccharides did not function as acceptors for the 1,3-fucosyltransferase.The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+ or Zn2+ ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected byN-ethylmaleimide.Abbreviations 1,3-Fuc-T GDP-fucose:-N-acetylglucosamine(Fuc to Asn-linked GlcNAc)1,3-fucosyltransferase - 1,6-Fuc-T GDP-fucose:-N-acetylglucosamine(Fuc to Asn-linked GlcNAc) 1,6-fucosyltransferase - PA pyridylamino - GnGn GlcNAc1-2Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4GlcNAc - GnGnF3 GlcNAc1-2Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-3)GlcNAc - GnGnF6 GlcNAc1-2-Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc - GnGnF3F6 GlcNAc1-2Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-3)[Fuc1-6]GlcNAc - MM Man1-6(Man1-3)Man1-4GlcNAc1-4GlcNAc - MMF3 Man1-6(Man1-3)Man1-4GlcNAc1-4(Fuc1-3)GlcNAc - MMF3F6 Man1-6(Man1-3)Man1-4GlcNAc1-4(Fuc1-3)[Fuc1-6]GlcNAc  相似文献   

9.
The release of the inhibitory amino acid -alanine was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The release was enhanced by -alanine itself and the structural analogs taurine and -aminobutyrate. It was dependent on Na+, but independent of Ca2+ in both mature and immature hippocampus, being thus mostly mediated by uptake carriers operating in an outward direction. The release was potentiated in the developing mice, but not affected in the adults, by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and tetrazolylglycine in a receptor-mediated manner. Cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals, greatly enhanced -alanine release at both ages, but more markedly in the adults. The great amounts of -alanine, together with the inhibitory amino acids taurine and -aminobutyrate, released simultaneously with the excitatory amino acids in the hippocampus may constitute an important protective mechanism against excitotoxicity, which leads to neuronal death.  相似文献   

10.
Volume-sensitive taurine transport in fish erythrocytes   总被引:5,自引:0,他引:5  
Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na+-dependent -amino-acid transport system which also required Cl for activity (apparentK m (taurine) 75 and 80 m;V max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na+/Cl/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na+-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na+-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.  相似文献   

11.
A statistical analysis of protein conformations in terms of the distance between residues, represented by their C atoms, is presented. We consider four factors that contribute to the determination of the distanced i,i+k between a given pair ofith and(i+k)th residues in the native conformation of a globular protein: (1) the distancek along the chain, (2) the size of the protein, (3) the conformational states of theith to(i+k)th residues, and (4) the amino acid types of the and(i+k)th residues. In order to account for the dependence on the distancek along the chain, the statistics are taken for three ranges, viz., short, medium, and long ranges (k8; 9k20; andk21; respectively). In the statistics of short-range distances, a mean distanceD k and its standard deviationS k are calculated for each value ofk, with and without taking into account the conformational states of all residues fromi toi+k (factors 1 and 3). As an Appendix, the relations for converting from the distances between residues into other conformational parameters are discussed. In the statistics of long-range distances, a reduced distanced* ij (the actual distance divided by the radius of gyration) is used to scale the data so that they become independent of protein size, and then a mean reduced distanceD l (a, a) and its standard deviation l (a, a) are calculated for each amino acid pair (a, a) (factors 2 and 4). The effect of the neighboring residues along the chain on the value of the distanced* ij is explored by a linear regression analysis between the actual reduced distanced* ij and the mean value over theD l for all possible pairs of residues in the two segments of the (i–2)th to the (i+2)th and the (j–2)th to the (j+2)th residues. The effect is assessed in terms of the tangentA l (a, a) of the calculated regression line for each amino acid pair (a, a). In the statistics of medium-range distances, only factors 1 and 4 are considered, to simplify the analysis. The scaled distanced i,i+k =(d i,i+k -D k )/S k is used to eliminate the dependence onk, the distance along the chain. The propertiesD m (a, a), m (a, a) andA m (a, a) corresponding toD l (a, a), l (a, a), andA l (a, a), and also calculated for each amino acid pair (a, a). The results are interpreted as follows: the smaller values ofD l (a, a) andD m (a, a) indicate a preference of the pair (a, a) for a contact (e.g., pairs between hydrophobic amino acids, and pairs of Cys with aromatic amino acids), and the larger values of these quantities indicate a preference for distant mutual location (e.g., pairs between strong hydrophilic amino acids); the smaller values of l (a, a) and m (a, a) indicate a strong preference for either contact or noncontact (e.g., pairs between hydrophobic amino acids, and pairs between strong hydrophobic and hydrophilic amino acids, respectively), and the larger values of these quantities indicate the ambivalent/neutral nature of the preference for contact and noncontact (e.g., pairs containing Ser or Thr); the smaller values ofA l (a, a) andA m (a, a) indicate that the distance of an (a, a) pair is determined independently of the amino acid character of the neighboring residues along the chain (e.g., some pairs of Cys or Met with other amino acids) and the larger values of these quantities indicare that such amino acid character contributes strongly to the determination of the distance (e.g., pairs containing Ser or Thr, and pairs between amino acids with small side chains). The difference between the statistics for the long- and medium-range distances is also discussed; the former reflect the difference between the hydrophobic and hydrophilic character of the residues, but the latter cannot be easily interpretable only in terms of hydrophobicity and hydrophilicity. The data analyzed here are used in the optimization of an object function to compute protein conformation in a subsequent paper.  相似文献   

12.
Luteinizing hormone beta (LH) and follicle stimulating hormone beta (FSH) subunits and their mRNAs were studied in the ram pars tuberalis following different seasonal (winter vs summer) and experimental (intact vs castrated animals) conditions. Hormone-containing cells were identified by immunohistochemistry, and mRNAs for LH and FSH by in situ hybridization using homologous double-stranded 35S-cDNAs. The labelling was quantified by image analysis. Immunohistochemical staining showed that cells containing LH and FSH were localized mainly in the ventral part of the pars tuberalis but that, in the summer, additional LH-containing cells were present in the dorsal part in intact rams. On the other hand, LH-mRNA labelling was found in the whole pars tuberalis in wethers but only in the ventral part in intact rams. The magnitude of LH-mRNA labelling was significantly greater in summer than in winter rams, and in castrated than in intact animals (P<0.001). However, the number of labelled cells was found to be the greatest in the winter (P<0.001) and was not affected by castration. FSH-mRNA expression was similar to that of LH-mRNA except that the level and extent were considerably lower. Thus, our results show an increase in the magnitude of gonadotropin subunit-mRNA in the summer and following castration; this increase appears to involve the entire pars tuberalis.  相似文献   

13.
Sequestered actin in chick embryo fibroblasts   总被引:1,自引:0,他引:1  
Chick embryo fibroblasts contain about 75-100 M unpolymerized actin and at least four proteins which can bind actin monomers, actin depolymerizing factor (ADF), gelsolin, profilin, and thymosin 4 (T4). Fibroblast extracts are analyzed by non-denaturing polyacrylamide gel electrophoresis and immunoblotting where most of the G-actin is detected as a complex with T4. When fibroblast extracts are fractionated by gel filtration and the fractions are analyzed by PAGE and HPLC, most of the G-actin elutes in a peak that also contains T4 at an overall molar ratio of 1.9:1 relative to actin. Gelsolin, profilin, and ADF are also detectable in the gel filtration eluate and at least partly coelute with actin, and account for only a minor fraction of the soluble actin pool. These observations indicate that under the growth conditions studied, T4 is the major actin-sequestering protein in fibroblasts.  相似文献   

14.
Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)2 from 13C R1 and R1 measurements on the C1, C3, and C4 carbons in samples uniformly enriched in 13C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R1/R1 ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R1/R1 ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D||/D, of 2.1±0.4, and an overall rotational correlation time, (2D||+4D)–1, of 3.35 ns at 35 °C in D2O, in excellent agreement with values obtained from hydrodynamic modeling.  相似文献   

15.
Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N2 fixation or supplied with NO3. The hydroponic medium was maintained at pH 7.5 by addition of CaCO3. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N2 fixation or supplied with NO3, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO3-supplied alfalfa was obtained at 100 M while maximum yield of N2-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N2 fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO3-grown and N2-fixing alfalfa were obtained at 10 M. Growth of NO3-supplied plants was inhibited at 200 M FeHEDTA while growth of N2-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N2 fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N2 fixation is more sensitive to limited Fe availability than alfalfa supplied with NO3.  相似文献   

16.
Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass–1 x h–1 were obtained in a 0.1 M Na2CO3/NaHCO3-buffer in a strirred bioreactor.  相似文献   

17.
Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and ethanol as carbon source. Growth on ethanol as the sole carbon source was only possible after the addition of a small amount of glutamic acid. The flows of glucose, ethanol, oxygen, carbon dioxide and biomass to and from the system were measured and a model for the growth of the yeast on the carbon sources constructed. The model is shown to allow independent estimation of YATP and P/O. YATP is not independent of the substrate used, but the amount of ATP used in the production of biomass from the monomers is approximately the same for growth on ethanol and on glucose.Nomenclature C chemical state vector - Ci component of the chemical state vector (C-mol) - Cx biomass present in the system (C-mol biomass) - H2 reduction equivalents (NAD(P)H + H+ and FADH2) - k the amount of ATP required in the production of 1 C-mol of biomass from the monomers (mol ATP/C-mol biomass) - mATP maintenance requirement for ATP (mol ATP/C-mol biomass·h) - P/O (=), efficiency of the oxidative phosphorylation (mol ATP/atom O) - r vector of reaction rates - ri component of the vector of reaction rates (C-mol/h) - rATP rate of ATP production (mol ATP/h) - rx rate of biomass production (C-mol biomass/h) - YATP YATP growth yield on ATP (C-mol biomass/mol ATP) - (YATP)max maximum growth yield on ATP - stoichiometry matrix - P/O - vector of the flows to the system - s flow of glucose to the system (C-mol glucose/h) - o flow of oxygen to the system (mol O2/h) - c flow of carbon dioxide to the system (mol CO2/h) - x flow of biomass to the system (C-mol biomass/h) - e flow of ethanol to the system (C-mol ethanol/h) - w flow of water produced during metabolism (mol H2O/h)  相似文献   

18.
Some of the characteristics of unisite hydrolysis of [32P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [32P]ATP bound at the catalytic sites of F1 under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [32P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [32P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [32P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [32P]ATP/32Pi bound at the catalytic site and a 50% diminution in the rate of 32Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F1 in the sense that in a fraction of F1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [32P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.  相似文献   

19.
Transducin (T), a guanine nucleotide binding regulatory protein composed of -, -, and -subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the - and -subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.  相似文献   

20.
A ouabain sensitive inward current occurs in Xenopus oocytes in Na+ and K+ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pHo) produces a conducting pathway through the Na+/K+ pump that is permeable to H+ and blocked by [Na+]o. An alternative suggestion is that the current is mediated by an electrogenic H+-ATPase. Here we investigate the effect of pHo and [Na+]o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pHo the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pHo, as predicted by a model in which protonation of the Na+/K+ pump reduces the energy barrier between the internal solution and the Na+ occluded state. The model also predicts that acidic pHo increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. 22Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pHo suggest that both Na+ and H+ are permeant. The acid-induced inward current is reduced by high [Na+]o, consistent with block by Na+. A least squares analysis predicts that H+ is four orders of magnitude more permeant than Na+, and that block occurs when 3 Na+ ions occupy a low affinity binding site (K 0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na+ and H+ through the Na+/K+ pump.  相似文献   

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