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S Joshi

Publications and source records attributed to S Joshi.

At least 217 records · Page 12Linked to original sources
PubMed1985

Resolution and reconstitution of H+ -ATPase complex from beef heart mitochondria.

Mitochondrial H+ -ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a "membrane" (NaBr-F0) and a "soluble" fraction by treatment with 3.5 M sodium bromide. The NaBr-F0 fraction is completely devoid of beta, delta, and epsilon subunits of the F, ATPase and largely devoid of alpha and gamma subunits of F1, where F0 is used to denote the membrane fraction and F1, coupling factor 1. This is confirmed by complete loss of ATPase and Pi-ATP exchange activities. The addition of F1 (400 micrograms X mg-1 F0) results in complete restoration of oligomycin sensitivity without any reduction in the F1-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F1-F0 complex consequent to sodium bromide extraction. Restoration of Pi-ATP exchange and H+ -pumping activities require coupling factor B in addition to F1-ATPase. The oligomycin-sensitive ATPase and 32Pi-ATP exchange activities in reconstituted F1-F0 have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F1-F0 complex. The data suggest that the altered properties of NaBr-F0 observed in other laboratories are probably inherent to their F1-F0 preparations rather than to sodium bromide treatment itself. The H+ -ATPase (F1-F0) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35-37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F1, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler- and oligomycin-insensitive. The membrane-bound hydrophobic component F0, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and Pi-ATP exchange activities on binding to F1-ATPase (33). The purest preparations of bovine heart mitochondrial F0 show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial F0 is not known. The F0 preparations from bovine heart reported so far have been derived from H+ -ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1984

Identification of the 29,000-dalton protein and its relevance to oligomycin-sensitive 32Pi-ATP exchange in bovine heart electron transport particles.

There have been several reports on the involvement of a 29,000-dalton protein in the regulation of ATP synthesis and 32Pi-ATP exchange (Zimmer, G., Mainka, L., and Heil, B. M. (1982) FEBS Lett. 150, 207-210). The present communication demonstrates that incubation of electron transport particles with 50 microM copper-o-phenanthroline results in reversible loss of 32Pi-ATP exchange but not of oligomycin-sensitive ATPase. Dependence of the inhibition on oxygen, its prevention by EDTA, ATP, or 2-mercaptoethanol, and subsequent restoration of the activity by 2-mercaptoethanol point to a thiol-disulfide interchange as the cause of inhibition. Analysis of copper-o-phenanthroline-treated samples by polyacrylamide gel electrophoresis conducted under nonreducing conditions shows four major changes. There is a decrease in the staining intensity of two bands with molecular weights of 34,000 and 29,000 with concomitant appearance of two new bands with molecular weights of 28,000 and 58,000-60,000. The 34,000-dalton band is tentatively identified as the phosphate transport protein. The 28,000-dalton component is formed by intramolecular and the 58,000-60,000-dalton component by intermolecular cross-linking of the 29,000-dalton protein. Pretreatment of electron transport particles with 2 mM N-ethylmaleimide does not affect 32Pi-ATP exchange or its inhibition by copper-o-phenanthroline but prevents cross-linking of the 34,000- and 29,000-dalton proteins. Evidence is presented to demonstrate that the purified H+-ATPase preparation has a single 29,000-dalton protein, identical to the adenine nucleotide translocase, and that it is not essential for 32Pi-ATP exchange or oligomycin-sensitive ATPase.

Adenosine Triphosphatases↗
PubMed1984

Immunologically reactive multiple species of mitochondrial coupling factor B.

Earlier work had indicated that mitochondrial coupling factor B (FB) could be obtained with differing molecular weights, a highly active 13,000 form, a 29,200 form with low activity, and a partially purified 46,000 form with activity higher than the 29,200 form. We have analyzed FB preparations of different purity and after different types of treatment on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), followed by silver staining or immunostaining either with rabbit anti-FB serum or monoclonal FB antibody. Highly purified preparations which appear as single bands in SDS-PAGE develop additional higher molecular weight bands (silver staining), including a 48,000 and a 68,000 band, after lyophilization or repeated freezing and thawing or if subjected to SDS-PAGE in the absence of thiol compounds. FB prepared without addition of dithiothreitol and glycerol for stabilization also shows high molecular weight forms, although the active fractions are obtained consistently in the final gel filtration step of purification at a position corresponding to Mr = 13,000. When FB preparations are analyzed by immunoblots of SDS-PAGE using a monoclonal antibody to FB, fresh preparations of purified FB show a single band, while multiple bands are seen in samples which have been frozen and thawed repeatedly. Preparations made in the absence of dithiothreitol and glycerol also show cross-reactive forms of high molecular weight. Similar immunoblots using rabbit antiserum with mitochondria, its extracts, and partially purified FB preparations, all show the presence of several higher molecular weight forms. It is concluded that FB is probably a monomer in mitochondria, and it appears to undergo oligomerization after extraction and during purification.

Adenosine Triphosphatases↗
PubMed1984

Sequence of an antifreeze protein precursor.

The amino acid sequence of the precursor to the second-most-abundant serum antifreeze protein (B) in the winter flounder has been determined by a combination of protein and DNA sequencing. The precursor is an 82-residue preproprotein which differs in only three positions from the amino acid sequence of the precursor for the most-abundant serum antifreeze protein (A). The base changes responsible for these substitutions, as well as several silent changes, are all clustered within the DNA coding for the mature protein portion. Among the post-translational modifications that the precursor undergoes is the removal of the c-terminal glycine residue. Cloning of full-length antifreeze protein cDNA has enabled us to identify the transcription initiation site which occurs 49 nucleotides upstream from the initiation codon.

Amino Acid Sequence↗
PubMed1983

tRNA-like structures of plant viral RNAs: conformational requirements for adenylation and aminoacylation.

Bromo- and cucumovirus RNAs contain a tRNA-like structure as an integral part of their genome. This structure is located at the 3' end of the viral RNA and is an acceptor of tyrosine. The 3' regions of representative viral RNAs have been sequenced and quite unorthodox secondary foldings have been proposed for these 3' ends. The question therefore remained as to how these structures could be recognized by tRNA-specific enzymes. We have established the minimum number of nucleotides from the 3' end of the brome mosaic virus and broad bean mottle virus RNAs required for the formation of structures recognized by the tyrosyl-tRNA synthetase and/or the tRNA nucleotidyltransferase. The results obtained delineate the length of the tRNA-like region, and indicate that the 5' region of the tRNA-like structure participates in the formation of the amino acid stem. This has led us to propose an 'L'-shaped secondary structure for these tRNA-like regions.

Anticodon↗
PubMed1982

On the role of factor B and oligomycin on generation and discharge of the proton gradient.

Bovine mitochondrial coupling factor B (FB) stimulates oxidative phosphorylation as well as other energy-linked reactions which are supported by ATP hydrolysis. Extraction of FB from submitochondrial particles results in a decrease in ATP-dependent proton translocation (delta pH) and binding of the voltage-sensitive dye, oxonol VI. Reconstitution of deficient particles with FB restores ATP-dependent proton translocation and oxonol binding but has little effect on oxonol binding supported by respiratory substrates.l In contrast, low levels of oligomycin stimulate oxonol binding supported by either ATP or NADH. In the case of oligomycin, the stimulatory effect is associated with a decrease in the rate of discharge of the proton gradient, but the depletion of FB or its restoration has no detectable effect on the discharge rate. The data indicate that the primary effect of FB is upon the rate of H+ translocation coupled to ATP hydrolysis, while that of low oligomycin is on the H+ permeability of the membrane.

Animals↗
PubMed1982

Length requirements for tRNA-specific enzymes and cleavage specificity at the 3' end of turnip yellow mosaic virus RNA.

This paper describes the minimum length of the turnip yellow mosaic virus (TYMV) RNA necessary to fulfill the tRNA-like properties of the viral RNA: 50 to 75 nucleotides and 86 nucleotides from the 3' end of TYMV RNA are sufficient for adenylation and valylation respectively by the Escherichia coli system. The size of the tRNA-like fragments obtained in vitro in the presence of an E. coli, a reticulocyte or a chinese cabbage leaf extract has also been determined. Among the major fragments liberated from the 3' end of TYMV RNA by the three systems are fragments of 117 and 112 nucleotides. In addition, the E. coli extract liberates fragments of 139 and 61 nucleotides, and the reticulocyte lysate fragments of 109, 94, 84, 73 and 46 nucleotides. The cleavage of the viral RNA by several systems in vitro to yield RNA fragments encompassing the tRNA-like sequence suggests that such fragments might also be liberated in vivo.

Base Composition↗
PubMed1982

Turnip yellow mosaic virus RNA is aminoacylated in vivo in Chinese cabbage leaves.

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure as an integral part of its genome. This structure is located at the extreme 3' end of the viral RNA and is the acceptor of valine after 3'-terminal adenylation. It is known that in vitro (with bacterial, yeast, or plant systems) and in vivo (upon microinjection into Xenopus laevis oocytes) a series of tRNA-specific enzymes can recognize this structure in the viral RNA. We report that TYMV RNA is valylated and consequently adenylated in vivo in its natural host, Chinese cabbage leaves. This suggests that the acylated form of the viral RNA could play an important role in the life-cycle of the virus.

Journal Article↗
PubMed1982

Isolation of a highly active H+-ATPase from beef heart mitochondria.

The lysolecithin extraction procedure originally described by Sadler et al. (1974) has been modified to yield a H+-ATPase with high levels of Pi-ATP exchange activity (400-600 nmol x min-1 x mg-1). This activity is further enhanced (1400-1600 nmol x min-1 x mg-1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H+ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. Pi-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).

Adenosine Triphosphatases↗
PubMed1981

Inhibition of coupling factor B activity by cadmium ion, arsenite-2,3-dimercaptopropanol, and phenylarsine oxide, and preferential reactivation by dithiols.

Coupling factor B activity was measured by the stimulation of the ATP-driven NAD+ reduction by succinate or the 32Pi-ATP exchange activity of Factor B-depleted submitochondrial particles. Half-maximal coupling activity was inhibited by 30 microM cadmium, 5 microM phenylarsine oxide, or 0.3 mM arsenite-2,3-dimercaptopropanol. The inhibition was relieved by slight excess of dithiol but not by a 10-fold molar excess of 2-mercaptoethanol. Inhibition of coupling activity by phenylarsine oxide or cadmium was not due to interference in binding of Factor B to depleted particles. Isolated Factor B binds phenylarsine oxide resulting in loss of ability to stimulate depleted submitochondrial particles. The inhibition was largely overcome by dithiol but not by monothiols. The residual coupling activity of depleted submitochondrial particles was highly resistant to cadmium or arsenical. Moreover, binding of arsenical to the depleted particles per se, did not result in inhibition of Factor B-stimulated activity. Furthermore, the addition of phenylarsine oxide to H+-ATPase resulted in loss of Pi-ATP exchange and stimulation of oligomycin-sensitive ATPase activities. Both effects were further potentiated by 2-mercaptoethanol and reversed by dithiols. These effects parallel uncoupling of oxidative phosphorylation in mitochondria by these inhibitors and point to Factor B as the probable component sensitive to these inhibitors.

Adenosine Triphosphatases↗
PubMed1981

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations.

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient sub-mitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations. The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor. Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F1 (0.3--0.6 nmol/mg). Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3. Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle.

Animals↗