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1.
Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either 3H- or 14C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of 14C3H ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the 14C3H incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.  相似文献   

2.
Joël Lunardi  Pierre V. Vignais 《BBA》1982,682(1):124-134
(1) N-4-Azido-2-nitrophenyl-γ-[3H]aminobutyryl-AdoPP[NH]P(NAP4-AdoPP[NH]P) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of 3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F1) was studied in the absence of photoirradiation, and compared to that of [3H]AdoPP[NH]P. The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once [3H]NAP4-AdoPP[NH]P had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by [3H]NAP4-AdoPP[NH]P upon photoirradiation. (3) Photoirradiation of F1 by [3H]NAP4-AdoPP[NH]P resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol [3H]NAP4-AdoPP[NH]Pmol F1. Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound [3H]NAP4-AdoPP[NH]P was localized on the α- and β-subunits of F1. At low concentrations (less than 10 μM), bound [3H]NAP4-AdoPP[NH]P was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F1.  相似文献   

3.
Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with N-ethyl[1?14C]maleimide, phenyl[203Hg]mercuric acetate and p-chloro[203Hg]mercuribenzoate showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with 14C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.  相似文献   

4.
Using guanidinium and n-butylammonium cations (C+) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H2A, three association complexes may exist: K1M = [CHA][C+] [HA?]; K1D = [CA?][C+] [A2?]; K2D = [C2A][C+] [CA?]. For guanidinium ion and phosphate, K1M = 1.4, K1D = 2.6, and K2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pKa = 4.8, K1M = 0.37; formate, pKa = 3.8, K1M = 0.32; and chloroacetate, pKa = 2.9, K1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.  相似文献   

5.
[S-[13C]methylmethionine-8 and -81]glycophorin A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the S-[13C]methylmethyionine-81 residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the S-[13C]methylmethionine-8 residue.  相似文献   

6.
Perfused rat hearts were treated with isoprenaline (10?6M) or ouabain (5.5 × 10?6M). The phosphate contents of troponin-I and myosin P light chains were established by radiolabelling with 32P; in the case of the light chains, direct chemical analysis of total and of specifically alkali-labile phosphate was also performed. Addition of isoprenaline caused phosphorylation of both troponin-I and myosin P light chains, reaching a maximum increment, after several minutes, of 1 mol/mol and 0.30 mol/mol, respectively. The Mg2+-ATPase activities, at saturating Ca2+ concentrations, of natural actomyosin isolated from treated hearts were significantly depressed, and an inverse correlation was established between the phosphate content of troponin-I and the Vmax[Ca2+] of this ATPase activity. The Ca2+ sensitivity of the Ca2+Mg2+-ATPase was also decreased. These changes were all reversed by an incubation permitting dephosphorylation of proteins by endogenous phosphatases.Treatment of hearts with ouabain caused no increment in troponin-I phosphorylation, but increased the P light chain phosphate content to a maximum of 0.30 mol/mol after some minutes. A positive correlation was evident between phosphate content of the light chains (in all experiments) and the maximum myosin Ca2+-ATPase activities. In addition, the Vmax[ATP] of the Ca2+Mg2+-ATPase of natural actomyosin was increased when light chain phosphorylation had occurred in the absence of troponin-I phosphorylation. P-light chain phosphorylation did not affect the Ca2+ sensitivity of Ca2+Mg2+-ATPase activity.We suggest that the effects of phosphorylation of troponin-I are to diminish thin filament sensitivity to Ca2+, and to decrease the efficiency of the transduction process along neighbouring actin monomers, such that the number of actin-myosin crossbridge interactions is decreased even in the presence of Ca2+ excess. Phosphorylation of P light chains of myosin has an activating effect on myosin Ca2+-ATPase activity, as well as on the rate of cross-bridge formation.  相似文献   

7.
Diploid embryos which are homozygous for the t12 mutation die at the morula stage. In the current studies, ova from heterozygous (+t12) females were fertilized in vitro with spermatozoa from +t12 males. The fertilized ova were immediately placed into media containing cytochalasin B to prevent second polar body formation, producing +/+/+, +/+/t12, +/t12/t12, and t12/t12/t12 embryos. The subsequent development of these triploid embryos was compared with that of diploid +/+, +t12, and t12t12 embryos developing from ova which were also fertilized in vitro with spermatozoa from +t12 males but which were not treated with cytochalasin B immediately following gamete coincubation. The data show that those triploid embryos which possess a wild-type allele and two mutant alleles are phenotypically wild type while those possessing three mutant alleles are not phenotypically distinguishable from their diploid (t12t12) counterparts. Like t12t12 embryos, t12/t12/t12 embryos die at the morula stage, prior to blastocoelic cavity formation.  相似文献   

8.
The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c has been measured as a function of the exogenous [ATP][ADP][Pi] ratio and pH. The respiratory rate at [ADP][ADP][Pi] values of less than 10-1m-1 is proportional to the reduction of cytochrome c and independent of pH from pH 6.5 to pH 8.O. The maximal turnover number (at 100% reduction) for cytochrome c is approximately 70 s?1. As the [ATP][ADP][Pi] ratio is increased from 10?1m?1 to 104m?1, the respiration at any given level of reduction of cytochrome c is progressively inhibited. Greater inhibition is observed at more oxidized levels of cytochorme c with respiratory control values for oxidation of reduced cytochrome c exceeding 10. The behavior of mitochondrial respiratory control is shown to be quantitatively consistent with a proposed mechanism in which the regulation occurs in the reaction of oxygen with cytochrome oxidase. A steady-state rate expression is derived which fits the mitochondrial respiratory rate dependence on (i) the extramitochondrial [ATP][ADP][Pi] ratio; (ii) the level of reduction of cytochrome c (or the intramitochondrial [NAD+][NADH]) at different [ATP][ADP][Pi] values; (iii) the pH of the suspending medium. This rate expression appears to correctly predict the relationships of the cytoplasmic [ATP][ADP][Pi] ratio, the mitochondrial [NAD+][NADH] ratio, and the mitochondrial respiratory rate in intact cells as well as suspensions of isolated mitochondria.  相似文献   

9.
[N-13CH3] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [N-13CH3] phosphatidylcholine is measured with 13C NMR. The transbilayer movements of [N-13CH3]-dioleoyl phosphatidylcholine and [N-13CH3] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [N-13CH3] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.  相似文献   

10.
Ultrastructural histochemistry instead of acrylamide gel electrophoresis (see R. Yasbin, J. Sawicki, and R. J. MacIntyre, 1978, Develop. Biol. 63, 00-00) is used to determine the time of paternal gene expression for the enzyme acid phosphatase-1 of Drosophila melanogaster in Acph-1nAcph-1B embryos in which the null allele is derived from the female parent. Timed embryos were histochemically stained for acid phosphatase activity according to the lead phosphate method of Gomori and were examined at the ultrastructural level. Enzyme activity, resulting from activation of the paternal Acph-1 gene, is detected as early as 5 hr after fertilization. Maternally derived enzyme in Acph-1BAcph-1B embryos is found principally in the yolk regions and around invaginations. This suggests that acid phosphatase-1 functions in yolk digestion and in cell movements during early embryogenesis.  相似文献   

11.
The observed equilibrium constants (Kobs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg2+]. Using Σ and square brackets to indicate total concentrations Kobs = Σ L-serine][Σ Pi]Σ L-phosphoserine]H2O], K = L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O]. The value of Kobs has been found to be relatively sensitive to pH. At 38 °C, K+] = 0.2 m and free [Mg2+] = 0; Kobs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔGobs0 = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H2O] = 1). The effect of the free [Mg2+] on Kobs was relatively slight; at pH 7.0 ([K+] = 0.2 m) Kobs = 52.0 m at free [Mg2+] = 10?3, m and 47.8 m at free [Mg2+] = 10?2, m. Kobs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔGobs0 = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H2O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 104, m [ΔGobs0 = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. Kobs = [Σ L-serine][Σ Pi][Σ L-phosphoserine][H2O], K = [L-H · serine±]HPO42?][L-H · phosphoserine2?]H2O. Values of Kobs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg2+], being calculated to be 668 [ΔGobs0 = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg2+] = 0; 111 [ΔGobs0 = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg2+] = 10?3, m; and 9.1 [ΔGobs0 = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg2+] = 10?2, m). Kobs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of Kobs are 4000 [ΔGobs0 = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg2+] = 0; and 97.4 [ΔGobs0 = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg2+] = 10?3, m. Combining Kobs for the l-phosphoserine phosphatase reaction with Kobs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of Kobs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. Kobs = [Σ L-serine][Σ NADH][Σ Pi][Σ α-ketoglutarate][Σ d-3-phosphoglycerate][Σ NAD+][Σ L-glutamat0] The value of Kobs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K+ as the monovalent cation) is 1.34 × 10?2, m at free [Mg2+] = 0 and 1.27 × 10?2, m at free [Mg2+] = 10?3, m.  相似文献   

12.
Rainbow trout were treated with β-naphthoflavone and the hepatic microsomal cytochrome P-450 solubilized with 3-[(3-cholaminopropyl)dimethylammonio]-1-propanesulfonate. Chromatography on tryptamine-Sepharose 4B gave a single cytochrome P-450 peak which was further resolved into three components by elution from DEAE-Sepharose. The two main peaks were then chromatographed on hydroxyapatite and a total of four fractions obtained. Two of these fractions had similar properties and significantly metabolized [14C]benzo[a]pyrene in a reconstituted system containing rat cytochrome P-450 reductase. This activity was inhibited by α-naphthoflavone but not by metyrapone or SKF-525A. Purified cytochromes P-448 from 3-methylcholanthrene-treated rat had similar spectral properties and activity towards [14C]benzo[a]pyrene suggesting similarities between these forms.  相似文献   

13.
The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the [ATP][ADP] ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the [NADH][NAD+] ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD+ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the [ATP][ADP] ratio brought about by glucagon may be secondary to the increase in the [NADH][NAD+] ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the [NADH][NAD+] and [ATP][ADP] ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD+ system.  相似文献   

14.
Hartmut Wohlrab  James Greaney 《BBA》1978,503(3):425-436
Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by N-[3H]ethylmaleimide, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the N-[3H]ethylmaleimidelabeled mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 103. The nmol N-[3H]-ethylmaleimide bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol N-[3H]ethylmaleimide/mol cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to N-[3H]ethylmaleimide and identified five labeled proteins. Only the labeled 32 · 103 dalton and the 45 · 103 dalton proteins are common to both systems  相似文献   

15.
Commercial [5-14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated R-5-phospho-[5-14C]mevalonate by ion-exchange chromatography. The artifactual 14CO2 results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the 14CO2 and [14C]cholesterol produced by rat renal cortex slices incubated with purified [5-14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that R[5-14C]mevalonate is genuinely oxidized to 14CO2invitro, and that purification of substrate before its use is necessary. Production of 14CO2 and various [14C]lipids from purified [5-14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.  相似文献   

16.
In vitro, the accumulation and release of [methyl-3H]thymidine ([3H]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [3H]thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated [3H]thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the [3H]thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm [3H]thymidine in the medium, the choroid plexus accumulated [3H]thymidine against a concentration gradient. However, the majority of the [3H]thymidine within the choroid plexus was metabolized to [3H]thymidine nucleotides at low extracellular [3H]thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration.  相似文献   

17.
The oxygen dependence of cellular energy metabolism.   总被引:14,自引:0,他引:14  
Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μm with an apparent Km for oxygen of less than 1 μm. In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μm downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic [ATP][ADP][Pi] and intramitochondrial [NAD]+][NADH] because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and [ATP][ADP][Pi].  相似文献   

18.
Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[14C] glucosamine into N-acetyl[14C]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [14C]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[14C]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[14C]glucosamine-labeled glycoprotein reveals two N-acetyl[14C]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[14C]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[14C]chitobiose. The linkage between the 14C-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[14C]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[14C]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[14C]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[14C]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[14C]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[14C]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled 14C-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[14C]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the 14C-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [14C]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.  相似文献   

19.
Nt-Methylhistidine, a non-reutilised amino acid present in some myofibrillar proteins, was radioactively labelled in vito with [Me-3H]methionine. The specific radioactivities of protein-bound methylhistidine and free methylhistidine in perfusate after perfusion of rat hind limbs taken from prelabelled rats was determined. The decrease in urinary methylhistidine activity with time was determined for rats similarly labelled. Comparison of the specific activities of free and bound methylhistidine and the non-linear semilogarithmic plot of urinary methylhistidine activity suggest that the myofibrillar protein catabolism, as indicated by methylhistidine release, may not be a simple exponential process. The possibility of non-random decay is discussed and an alternative model proposed.  相似文献   

20.
Robert F. Anderson 《BBA》1983,723(1):78-82
The bimolecular decay rates (2k) of the flavosemiquinones (FH·F?) of riboflavin, FMN and FAD have been determined using pulse radiolysis. The rates (defined as d[FH·F?]dt = ?2k[FH·F?]2) for the neutral flavosemiquinones at zero ionic strength and pH 5.9 are (in units of mol?1·dm3·s?1): (1.2 ± 0.1)·109, (5.0 ± 0.2)·108 and (1.4 ± 0.1)·108; and for the anionic flavosemiquinones at pH 11.2 (5.4 ± 0.9)·108, (4.5 ± 0.3)·107 and (8.5 ± 1.3)·106, respectively. The kinetic salt effect has been used to formulate rate equations for each flavin to adjust for ionic strength effects.  相似文献   

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