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161.
Evolution of enzyme catalytic power. Characteristics of optimal catalysis evaluated for the simplest plausible kinetic model 总被引:3,自引:3,他引:0 下载免费PDF全文
Keith Brocklehurst 《The Biochemical journal》1977,163(1):111-116
1. Evolutionary changes in the structure of an enzyme that provide an increase in its Km value are considered. Provided that Km increases as a result of increases in the forward rate constants of the catalysis relative to the reverse rate constants, the enzyme catalyses the conversion of a fixed concentration of its substrate more rapidly when its structure provides that Km>[S] than when Km<[S]. 2. Catalytic efficiency of enzymes is discussed in terms of the simplest plausible model, the Haldane [(1930) Enzymes, Longmans, London] reversible three-step model: [Formula: see text] The rate equation for the forward reaction of this model (formation of P) may be written in the simple form: [Formula: see text] Keq. is the equilibrium constant (=[P]eq./[S]eq.), and kcat.=V/[E]T, where [E]T is the total enzyme concentration. 3. To assess the effectiveness of an enzyme, it is necessary only to determine the extent to which the constraints of a particular kinetic mechanism permit v2 (v when Km»[S]) to approach vd (the diffusion-limited rate). 4. The value of the optimal rate of catalysis (vopt., the maximal value of v2) is dictated by the equilibrium constant for the reaction, Keq.; v2=vd/a, where [Formula: see text] when k+1 is assumed equal to k−3, and vopt.=vd/amin.. When Keq.≥1, it is necessary that k+2»k−1 for a to take its minimum value, amin.; when Keq.«1, it is necessary only that k+2»Keq.·k−1, i.e. a can equal amin. even if k+2<k−1. When Keq.»1, vopt.=vd; when Keq.=1, vopt.=vd/2, and when Keq.«1, vopt.=Keq.·vd. 5. The analysis, together with predicted effects of evolutionary pressure, suggests that in practice the rates of the fastest enzyme-catalysed freely reversible reactions might be expected to be lower than the value of k+1[E]T[S] by about an order of magnitude, particularly if Keq.<1. 6. The existing literature suggests that, in general, appropriate values of Km have evolved for the provision of high rates of catalysis but that many values of kcat. are not large enough to provide optimal rates of catalysis unless the value of k+1 in vivo is lower than its value in free solution. 相似文献
162.
1. A method is described for the preparation of isolated cells from guinea pig liver. This involved perfusion in situ, in the non-physiological direction, with collagenase. 2. The cell yield was 20--30%, comparable with those from the livers of other species. 3. The ratio of lactate dehydrogenase to glutamate dehydrogenase in the cells was similar to that in vivo, indicating that there was negligible leakage of cytoplasmic enzymes. 4. The concentrations of K+ and adenine nucleotides were initially lower than in the perfused liver; normal values were obtained on incubation, particularly in the presence of substrate. 5. The L-lactate: pyruvate ratio is 16:1, close to established values. The total beta-hydroxybutyrate: acetoacetate ratio indicates that the mitochondrial redox state is more oxidised than in the perfused liver, but the intracellular ratio is similar to that of the intact liver. 6. Rates of gluconeogenesis and ureogenesis, are within the physiological range. Maximal gluconeogeneis from L-lactate was preceded by a lag period. L-lysine stimulated glucose production from L-lactate but did not abolish the lag phase. 7. The effects of aminooxyacetate and octanoate on L-lactate gluconeogenesis were similar to those in the perfused liver. 相似文献
163.
Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses 总被引:2,自引:1,他引:1 下载免费PDF全文
Bernard Moss Jerry M. Keith Alan Gershowitz Mary B. Ritchey Peter Palese 《Journal of virology》1978,25(1):312-318
Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-32P]guanosine 5′-triphosphate or S-adenosyl[methyl-3H]methionine to form “cap” structures of the type m7G(5′)pppNm-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m7G(5′)pppAm, m7G(5′)pppAmpGp, and m7G(5′)pppAmpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution. 相似文献
164.
The seed globulins of Lupinus angustifolius are glycoproteins containing 1.4–1.9% (α-conglutin), 2.8–6.4 % (β-conglutin) and 1.2–3.8% (γ-conglutin) carbohydrate. The highest values were obtained after acid hydrolysis and determination by phenol—H2SO4, (α, γ-conglutins) or by methanolysis and sugar determination by GLC (β-conglutin). TCA denaturation of β- and γ-conglutins was necessary to remove adsorbed galactomannans before determination of glycoprotein carbohydrates. All 3 conglutins contained mannose, galactose and glucosamine, though the ratio of mannose to galactose, and to a lesser extent neutral sugars to hexosamine varied. Small amounts of fucose were found associated only with γ-conglutin. 相似文献
165.
166.
The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41°C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive. 相似文献
167.
Continuous culture system for production of biopolymer levan using erwinia herbicola 总被引:1,自引:0,他引:1
Keith J Wiley B Ball D Arcidiacono S Zorfass D Mayer J Kaplan D 《Biotechnology and bioengineering》1991,38(5):557-560
The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer. 相似文献
168.
Keith Paige Melanie Palomares Patricia A. D’Amore Susan J. Braunhut 《In vitro cellular & developmental biology. Animal》1991,27(2):151-157
Summary The growth of the endothelial cell (EC) is tightly regulated throughout the body. Many factors have been implicated in modulating
EC growth including diffusible compounds, cell-to-cell interactions, and the extracellular matrix (ECM). Retinol, or vitamin
A alcohol, has recently been shown to inhibit the growth of bovine capillary ECs, in vitro. Retinoids are known to modify
ECM in other cell systems, and pure ECM components have been shown to effect EC growth rates. We, therefore, examined the
role of the matrix in the retinol-induced inhibition of ECs. Cell-free matrices from control and vitamin A-treated ECs were
prepared by removing cells with EGTA treatment after 7 d of culture. Matrix proteins were analyzed by solubilizing the matrices
in 5M quanidine-HCl and performing Western blot analysis using specific antibodies to matrix proteins. In isolating the ECM, we
observed that retinol-treated cultures of ECs were resistant to EGTA removal; retinol-treated ECs required twice the exposure
time to EGTA to detach from their matrix than did controls cells. Western blot analysis of matrix proteins derived from control
and retinol-treated EC cultures demonstrated a 1.6-fold increase in lamininβ chains and a 2.5-fold increase in fibronectin in the ECM of retinol-treated EC compared to control cell matrix. Functional
properties of these matrices were assessed by plating control and Day 6 retinol-treated ECs onto the matrices and measuring
attachment and growth by determining cell numbers at 24, 72, and 144 h. These studies revealed that control cells attached
in greatest numbers to a control matrix whereas retinol-treated ECs preferentially attached to a matrix derived from retinol-treated
cells. Furthermore, control ECs which grew rapidly on a control matrix were growth inhibited on a retinol-derived matrix.
These data indicate that vitamin A treatment of ECs effects both their phenotype and influences the composition and the functional
properties of their underlying ECM. These studies also demonstrate that alterations of the matrix are at least in part responsible
for the growth inhibition of EC by retinol. 相似文献
169.
Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process. 相似文献
170.
In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation. 相似文献