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121.
Partition kinetics of ribulose-1,5-bisphosphate carboxylase from Rhodospirillum rubrum 总被引:1,自引:0,他引:1
When the enzymatically generated intermediate 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate (II) was used as a substrate with fresh enzyme, 70% reacted to produce 3-phosphoglycerate (3PGA). When a reaction mixture of enzyme plus [1-32P]ribulose 1,5-bisphosphate (RuBP) was quenched in the steady state with the tightly bound inhibitor 2-carboxyarabinitol-1,5-bisphosphate, 30% of the enzyme-bound species was released as 3PGA and 70% as RuBP. The major source for this partition was the ternary substrates Michaelis complex. The level of carboxylated intermediate in the steady state was determined to be 8% of active sites under the conditions of substrate saturation. No burst was seen in the appearance of product when 6.5 eq of [1-32P]RuBP was mixed with enzyme plus saturating CO2 and the reaction followed in the steady state. From these data plus the steady-state Vmax and Km of RuBP it is possible to derive the five bulk rate constants represented in the scheme ECO2 + RuBP in equilibrium ERuBPCO2 in equilibrium E X II----E + 2(3PGA). 相似文献
122.
Samuel H. K. Ho Uttam Das Gupta John S. Rieske 《Journal of bioenergetics and biomembranes》1985,17(5):269-282
Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochromec reductase (Complex III) purified from bovine heart (K
i
=ca. 0.5 µM) was considerably less than that of antimycin (K
i
3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [3H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m=13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [3H]DAA, respectively. The labeling of band 7 by [3H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohybride or ascorbate. Based on magnitude of labeling by [3H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m=16 kDa) showing a lesser but significant amount of specific binding. 相似文献
123.
Whereas glucose is a major substrate for pulmonary lipid synthesis, fructose has also been suggested as a potential substrate. In vivo pulmonary fatty acid synthesis is depressed in hormonally deprived conditions, such as diabetes, and this can be modified by fructose feeding, but not by glucose feeding. In this study the glucose and fructose utilizations were compared in normal, diabetic and fasting states using isolated perfused rat lungs. When (U-14C)- or (5-3H)-glucose was used as substrate, glucose utilization by lung was reduced by 50% in both the fasting and diabetic animals compared to the normal controls. Using (U-14C)-glucose as substrate, the incorporation of (14C)-label in various metabolites of glucose was significantly depressed. For example, this reduction was 50% in lactate, pyruvate and CO2, 15% in ethanol-insoluble fraction, 65% in neutral lipids, 75% in phospholipids, 80% in fatty acid moiety, 40% in deacylated fraction and 10% in the polysaccharide fractions. Refeeding the fasted animals or insulin treatment to the diabetic animals restored these depressed (14C)-recoveries to the normal levels. Fructose utilization was less than 10% of glucose utilization, but remained unaffected by fasting and diabetic states. In addition, pulmonary hexokinase enzyme activity was lowered significantly in fasting and diabetic animals, whereas fructokinase enzyme activity was not altered. Despite the low rate of fructose utilization, these results suggest that fructose may serve as an alternative substrate for pulmonary phospholipid synthesis when glucose utilization is significantly depressed. 相似文献
124.
Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport 总被引:41,自引:0,他引:41
The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G) contains 20 uncharged and mostly hydrophobic amino acids. We created DNAs specifying G proteins with shortened transmembrane domains, by oligonucleotide-directed mutagenesis. Expression of these DNAs showed that G proteins containing 18, 16, or 14 amino acids of the original transmembrane domain assumed a transmembrane configuration and were transported to the cell surface. G proteins containing only 12 or 8 amino acids of this domain also spanned intracellular membranes, but their transport was blocked within a Golgi-like region in the cell. A G protein completely lacking the membrane-spanning domain accumulated in the endoplasmic reticulum and was secreted slowly. These experiments indicate that the size of the transmembrane domain is critical not only for membrane anchoring, but also for normal cell surface transport. 相似文献
125.
126.
S M Liang B Allet K Rose M Hirschi C M Liang D R Thatcher 《The Biochemical journal》1985,229(2):429-439
Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue). 相似文献
127.
Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein. 总被引:30,自引:2,他引:28
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The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed. 相似文献
128.
John J. Langone Chandra Das Robin Mainwaring William T. Shearer 《Molecular and cellular biochemistry》1985,65(2):159-170
Summary Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 1311-Fc fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 1311-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.Abbreviations SpA
protein A of Staphyloccus aureus
- VBS
EDTA gel, 0.0055 M veronal buffered saline containing 0.01 M EDTA and 0.1% gelatin, pH 7.4
- PBS
0.01 M phosphate buffered saline, pH 7.4 相似文献
129.
130.