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21.
Kimberly A. Aeling Kirsty A. Salmon José M. Laplaza Ling Li Jennifer R. Headman Alex H. Hutagalung Stephen Picataggio 《Journal of industrial microbiology & biotechnology》2012,39(11):1597-1604
We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5′-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a Km for xylose of 66.7?mM and a specific activity of 1.41?μmol/min/mg. In comparison, the native xylose isomerase was found to have a Km for xylose of 117.1?mM and a specific activity of 0.29?μmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation. 相似文献
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An isothermal titration calorimetric (ITC) method was developed to measure the kinetic parameters of ribonuclease A catalytic hydrolysis of cytidine 2',3'-cyclic monophosphate. Employing the inhibition of product as a probe, the K(m), K(i), k(c), and DeltaH(m) can be determined by two simple calorimetric measurements. First, the substrate was titrated into the cell containing high concentration of enzyme. The molar reaction heat was calculated from the titration peak area divided by substrate moles per titration, and the initial catalytic reaction rate in the presence of various concentrations of product can be calculated from the peak height and the molar reaction heat. From Michaelis-Menten function in the presence of inhibitors, the relationship between K(m) and K(i) can be obtained. Then, the dissociation constant, which is equal to K(i), was measured by a regular ITC experiment. Thus, K(m) and k(c) can be calculated. The method developed here can be applied in other enzyme catalytic systems with inhibitive products. 相似文献
24.
To determine the effects of ischemia-reperfusion (I/R) on alpha(1)-adrenergic-receptor (alpha(1)-AR) functions, alpha(1)-AR-mediated contraction, inositol phosphate (IP) accumulation, and alpha(1)-AR-G protein coupling were examined in the tail arteries of anesthetized rats after 60 min of ischemia and 60 min of reperfusion. The contractile response to norepinephrine (NE) was significantly increased after I/R, whereas the contractile response to KCl remained unchanged. This was accompanied by a 69% increase in NE-stimulated IP accumulation. Furthermore, receptor-stimulated coupling of alpha(1a)-AR to G alpha(q/11) proteins was increased, whereas the coupling of alpha(1b)-AR or alpha(1d)-AR to their G proteins was not altered by I/R. These changes in vascular alpha(1)-AR function occurred without concurrent alteration in expression levels of membrane alpha(1)-AR subtypes or in the associated G proteins. These data demonstrate that I/R increases alpha(1a)-AR-G(q/11) protein coupling and alpha(1)-AR-stimulated IP accumulation in the tail artery. The alterations in alpha(1)-AR signaling are associated with and may underlie the enhanced contractile response of the tail artery to adrenergic stimulation after I/R. 相似文献
25.
Kuo Huang Ling Bai Jiun Chen Yun Wen Peng Shih Chong Tsai Fu Chuo Peng Chung Kuang Yang 《Mycotoxin Research》1987,3(2):58-64
The chemical reaction of cleavaging territrem B to give 3,4,5-trimethoxy benzoic acid by alkaline hydrogen peroxide was investigated. The method was applied for confirmation of the chemical structure of the aromatic moiety of territrem A, A’, B, and B’. The physicochemical properties of the aromatic cleavage product of territrem Aindicated the structure as 3,4-methylendioxy, 5-methoxy benzoic acid (or 4-methoxy, 6-carboxy, 1, 3-benzodioxole). The experiment also gave the evidences that territrem A and A’, on the other hand territrem B and B’ have the identical aromatic moieties on their structures. 相似文献
26.
Chromogranin A-like proteins in the secretory granules of a protozoan, Paramecium tetraurelia 总被引:2,自引:0,他引:2
J B Peterson D L Nelson E Ling R H Angeletti 《The Journal of biological chemistry》1987,262(36):17264-17267
The ciliate protozoan Paramecium tetraurelia produces secretory granules (trichocysts) which release needle-like structures composed of small, acidic proteins. Using antibodies against isolated chromogranin A (CGA) and against trichocyst proteins, we found cross-reactive proteins in chromaffin granules and trichocysts. Four independently derived sera against isolated CGA stained bands of the Mr 15,000-25,000 family of trichocyst proteins on immunoblots. A positive response was also obtained with antiserum against chemically synthesized peptides (PL26 and GE25) corresponding to defined regions of the CGA amino acid sequence. In extracts of whole Paramecium, larger proteins (Mr 53,000 and 49,000) also reacted with antibodies against CGA and the related synthetic peptides. These larger proteins may represent unprocessed precursors to the smaller proteins of mature trichocysts. Antiserum to trichocysts recognized CGA in chromaffin granule lysates. Further evidence of a Paramecium protein related to CGA was provided by hybridization of Paramecium mRNA with cloned cDNA for bovine CGA. Our results suggest striking conservation in evolution of CGA-like proteins that may play some role, as yet unknown, in secretion. 相似文献
27.
28.
Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins 总被引:7,自引:0,他引:7
F S Esch S Shimasaki K Cooksey M Mercado A J Mason S Y Ying N Ueno N Ling 《Molecular endocrinology (Baltimore, Md.)》1987,1(5):388-396
Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins. 相似文献
29.
A radioimmunoassay for human pro-luteinizing hormone-releasing factor [pro-LRF(14-69)OH] 总被引:1,自引:0,他引:1
A Motoyama I Wakabayashi S Minami H Sugihara F Takahashi S Akira N Ling 《Endocrinologia japonica》1987,34(1):133-137
A radioimmunoassay (RIA) for human pro-LRF(14-69)OH was developed with an antiserum, generated in a rabbit, to [Tyr67]pro-LRF(47-67)NH2 conjugated to BSA. This antiserum bound 28-32% of [125I]pro-LRF(14-69)OH at a final dilution of 1:2500 and the binding was inhibited by pro-LRF(14-69)OH in a dose-dependent manner. The sensitivity of the RIA was 31.2-62.5 pg and the dose that inhibited 50% of the binding to the tracer was 280-320 pg. Intra- and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. Neither LRF nor pro-LRF(14-37)OH was recognized by the antiserum. The dilution curve generated with human hypothalamic extract was parallel to that of pro-LRF(14-69)OH. In addition the extract yielded a major immunoreactive peak emerging in elution volumes concordant with [125I]pro-LRF(14-69)OH on Sephadex G-50 chromatography. 相似文献
30.