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41.
The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed. 相似文献
42.
Distribution and fetomaternal transfer of 6 beta-hydroxycortisol (6 beta-OHF) was studied using serial sampling following injection of tritium labelled 6 beta-OHF into various fluid compartments in the chronically cannulated unaesthesized pregnant ewe. There was a rapid transfer of 6 beta-OHF from the fetal circulation into amniotic fluid and maternal blood. In contrast, the maternal----fetal transfer of this steroid metabolite was considerably less. The sequence of appearance of 6 beta-OHF in fetal blood and amniotic fluid following injection into maternal blood suggests that this steroid is first transferred across the placenta to fetal blood before gaining entry into the amniotic fluid space. The half-lives of 6 beta-OHF after initial equilibration in maternal plasma, fetal plasma and amniotic fluid were 2.0 h, 5.1 h and 8.9 h respectively. The amniotic sac appears to contain a relatively static pool of 6 beta-OHF and may act as a "trap" for 6 beta-OHF in the ovine conceptus. 相似文献
43.
Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit 总被引:4,自引:0,他引:4
T Berg J Sulner C Y Lai R L Soffer 《The journal of histochemistry and cytochemistry》1986,34(6):753-760
The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme. 相似文献
44.
W. Conrad Liles Stephen Taylor Richard Finnell Henry Lai Neil M. Nathanson 《Journal of neurochemistry》1986,46(3):977-982
The tottering mouse (tg/tg) is a single-locus mutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalographic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tg/tg) and coisogenic wild-type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age-matched control wild-type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40-60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density. 相似文献
45.
The structural organization of the chicken calmodulin gene 总被引:13,自引:0,他引:13
R C Simmen T Tanaka K F Ts'ui J A Putkey M J Scott E C Lai A R Means 《The Journal of biological chemistry》1985,260(2):907-912
46.
Cholera toxin A subunit: functional sites correlated with regions of secondary structure 总被引:1,自引:0,他引:1
The A subunit of cholera toxin contains the ADP-ribosyltransferase activity in its major constituent polypeptide A1 (Mr 23,000) which is responsible for the elevation of cAMP typically observed with most mammalian cell types after exposure to the toxin. The primary structure of the A subunit, recently established by sequence analyses, is presented and used as the basis for the secondary structure prediction according to the method of Chou and Fasman. The results indicated the presence of 27% alpha-helix, 25% beta-structure, 12% beta-turn, and 36% random coil. The majority of the beta-structure consisted of six strands located in the NH2-terminal portion of the molecule (residues 33-106) covering one-half of the region corresponding to the A1 polypeptide portion. The beta-sheet domain led immediately into the active site region characterized by the alternating structures of beta-pleated sheet and alpha-helix (residues 95-140) similar to that reported for other NAD+ binding proteins. The presence of this structural feature in the region was confirmed by the use of another predictive method (J. Garnier et al., J. Mol. Biol. 1978, 120, 97-120). In addition, two regions (residues 14-18 and 200-214), previously identified to contain binding sites for the B subunit as evidenced by chemical modification and monoclonal antibody studies, were found to be in alpha-helix configuration. 相似文献
47.
Ca2+/calmodulin-dependent protein kinase II. Isozymic forms from rat forebrain and cerebellum 总被引:33,自引:0,他引:33
Ca2+/calmodulin-dependent protein kinase II, an abundant brain protein proposed to mediate a number of Ca2+-regulated processes in neuronal tissue, is composed of autophosphorylatable subunits of Mr 50,000 and 60,000/58,000. A recent study (McGuinness, T. L., Lai, Y., Greengard, P., Woodgett, J.R., and Cohen, P. (1983) FEBS Lett. 163, 329-334) suggested that this kinase exists as isozymes which vary in the relative ratio of these subunits in different tissues or species. Other studies (Walaas, S. I., Nairn, A. C., and Greengard, P. (1983) J. Neurosci. 3, 291-301, 302-311) provided evidence which suggested that the ratio of these phosphopeptides might vary in different brain regions. In the present investigation, we have tested this possibility by comparing Ca2+/calmodulin-dependent protein kinase II purified from rat forebrain and cerebellum. The two kinases had similar purification characteristics, subunit compositions, physical properties, and substrate specificities. Gel filtration and sucrose density gradient centrifugation provided an estimated molecular weight of 550,000 for the forebrain kinase and 615,000 for the cerebellar kinase. The kinases from the two regions clearly differed in the relative proportions of the Mr 50,000 and 60,000/58,000 subunits. Three independent methods indicated that the forebrain kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 3:1 ratio, while the cerebellar kinase contained the Mr 50,000/(60,000/58,000) subunits in approximately a 1:4 ratio. The forebrain kinase subunits were shown to be identical to the corresponding subunits of the cerebellar kinase by several criteria. The data are consistent with the existence in various brain regions of isozymic forms of Ca2+/calmodulin-dependent protein kinase II which differ in their relative subunit ratios. 相似文献
48.
Kwan-Fu Rex Sheu James C. K. Lai Young Tai Kim† Gary Dorante Jennifer Bagg 《Journal of neurochemistry》1985,44(2):593-599
Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
49.
Both positive and negative biological effects of microwaves on drug actions in rats exposed to 1-mW/cm2, 2,450-MHz microwaves have been reported by several investigators. We conducted dosimetry studies for seven different exposure conditions to determine whether these different results could be due to the rats having been exposed differently. They included anterior and posterior exposures in a circular waveguide, near field, far field with E- or H-field parallel to the long axis of the body and dorsal exposure in a miniature anechoic chamber with E- or H-field parallel to the long axis of the body. The average specific absorption rates (SARs) in the head, tail, and body of the exposed rats were measured by means of a calorimetry system. The local SARs at eight locations in the brain were determined by temperature measurement with Vitek probes. Intensive coupling of energy to the tail when it was exposed parallel to the E-field was shown by thermography. For the same average incident power density, the average SARs in the heads of rats were about two times higher in the circular waveguide than for other exposures. The local SARs in the brain varied for different exposure conditions. Statistical comparisons of SARs under the different exposure conditions are presented. 相似文献
50.
The nucleotide sequence of a rat liver glutathione S-transferase subunit cDNA clone 总被引:17,自引:0,他引:17
We have determined the nucleotide sequence of a cloned cDNA derived from liver poly(A) RNA of pentobarbital-treated rats encoding a glutathione S-transferase subunit. This cDNA clone pGTR261 contains one open reading frame of 222 amino acids, a complete 3' noncoding region, and 63 nucleotides in the 5' noncoding region. The cloned DNA hybridizes to rat poly(A) RNA in a tissue-specific fashion, with strong signals to liver and kidney poly(A) RNA(s) of approximately 1100 and approximately 1400 nucleotides in size but little or no hybridization to poly(A) RNAs from heart, lung, seminal vesicles, spleen, or testis under stringent conditions. Our sequence covers the cDNA sequence of pGST94 which contains a partial coding sequence for a liver glutathione S-transferase subunit of Ya size. Comparison of sequences with our earlier clone pGTR112 suggests that there are at least two mRNA species coding for two different subunits of the Ya (Mr = 25,600) subunit family with very limited amino acid substitutions mainly of conserved polarity. The divergent 3' noncoding sequences should be useful molecular probes in differentiating these two different but otherwise very similar subunits in induction and genomic structure analyses. Our results suggest that tissue-specific expression of the glutathione S-transferase subunits represented by the sequences of pGTR261 and pGTR112 may occur at or prior to the level of RNA processing. 相似文献