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D K Stone

Publications and source records attributed to D K Stone.

At least 37 records · Page 2Linked to original sources
PubMed1991

Vacuolar acidification and bafilomycin-sensitive proton translocating ATPase in human epidermal Langerhans cells.

Langerhans cells (LC) are the principal antigen-presenting cells (APC) of squamous epithelia. We have previously shown that freshly isolated LC (fLC) are able to deliver endocytosed membrane MHC class II molecules into acidic environments, and that this capacity is lost when LC are placed in culture (cLC). Inasmuch as processing of antigens requires their passage through acidic compartments, we undertook the present study to examine the ability of fLC and cLC to take up acridine orange, and to identify proton-translocating ATPases in these cells. Using flow cytometry and fluorescence microscopy, acridine orange was observed to accumulate in acidic compartments in both fLC and cLC. Using a radioactive ATPase assay, crude membrane preparations from both fLC and cLC were shown to possess three types of ion-translocating ATPase, based on sensitivity to the following inhibitors: ouabain (Na+, K+ ATPase), oligomycin (mitochondrial F1F0 ATPase), and bafilomycin (vacuolar-type proton pump ATPase); the last type is responsible for acidification in vacuolar compartments. cLC displayed markedly less (less than 50%) total ATPase activity compared to fLC; however, the relative proportions of specific ATPases were similar in fLC and cLC. Combined use of the three inhibitors resulted in abrogation of only 25-40% of the total ATPase activity. Finally, treatment of LC with bafilomycin inhibited both acridine orange uptake and acidification of internalized HLA-DR molecules. These results confirm the ability of both fLC and cLC to acidify vacuolar compartments, thereby suggesting that lack of acidification of endocytosed membrane class II molecules in cultured cells is due to alternative routing to non-acidic organelles.

Acridine Orange↗
PubMed1990

Structure of vacuolar proton pumps.

At the present time, knowledge of the structure of the proton-translocating ATPase responsible for urinary acidification is far from complete. Key issues awaiting resolution are full definition of subunit structure and an understanding of the interactions among functional domains of this complex hetero-oligomer. Such a detailed analysis is required to begin study of the biogenesis of the vacuolar proton pump and to elucidate the complexities of its molecular regulation.

Acid-Base Equilibrium↗
PubMed1989

Isolation and reconstitution of the chloride transporter of clathrin-coated vesicles.

Clathrin-coated vesicle acidification is mediated by an endomembrane proton translocating ATPase. This pump is electrogenic, and significant pH gradient formation requires the parallel movement of chloride through a chloride transporter in order to maintain net electroneutrality. We have solubilized, isolated and achieved 270-fold purification of this chloride transporter by means of selective detergent solubilization with cholate and polyoxyethelene 9-lauryl ether (C12E9), hydroxylapatite chromatography, and glycerol gradient centrifugation. Stabilization of the solubilized transporter requires 5 mM dithiothreitol. The partially purified transporter was co-reconstituted with the purified clathrin-coated vesicle proton translocating complex to yield preparations of proteoliposomes capable of valinomycin-independent proton pumping, as assessed by ATP-generated acridine orange quenching. In addition, the chloride transporter was independently reconstituted and was shown to catalyze diisothiocyano-disulfonic acid stilbene-sensitive 36Cl uptake. The anionic conductive selectivity of the reconstituted transporter (chloride = bromide greater than nitrate) exactly matched that of the transporter of native clathrin-coated vesicles. These studies demonstrate that the chloride transporter of vacuolar acidification systems is structurally and functionally dissociable from co-existing proton pumps and allow for investigations of pump-transporter interactions in a resolved system.

Animals↗
PubMed1989

Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain.

Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X. S., and Stone, D. K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 mumol of Pi.mg of protein-1.min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.

Affinity Labels↗
PubMed1989

Vacuolar proton pumps.

Recently a new class of proton-translocating ATPases has been localized to endomembrane compartments in plant, fungal, and mammalian cells. These proton pumps are large hetero-oligomers which have an ATP hydrolytic sector that is functionally and structurally distinct from a transmembranous proton pore. Enzymatic characteristics of these proton pumps are discussed as well as the current state of knowledge regarding subunit composition and function. In addition, recent primary sequence data are discussed which indicate that these proton pumps share a common ancestor with F1F0-type proton pumps of mitochondria.

Animals↗
PubMed1989

Human endomembrane H+ pump strongly resembles the ATP-synthetase of Archaebacteria.

Preparations of mammalian H+ pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H+ pump are unknown from animal cells. We report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H+ pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H+ gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H+ pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

Amino Acid Sequence↗
PubMed1988

Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis.

The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S.Z., Xie, X. S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase-and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3-, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1988

Structural properties of the proton translocating complex of the clathrin-coated vesicle.

The clathrin-coated vesicle proton pump is a representative member of the new class of endomembrane proton ATPases that share an inhibitor profile which distinguishes them from classic F1F0 and E1E2-type proton pumps. The coated vesicle proton pump is a large (530 kDa) heteroligomer composed of eight polypeptides with molecular masses of 116, 70, 58, 40, 38, 34, 33 and 17 kDa. The 200-fold purified enzyme catalyses ATP-generated proton pumping when reconstituted in liposomes composed of pure lipids. Subunit function has been determined by partial reaction analysis of subunit and subcomplex activities. The isolated 17 kDa subunit, when co-reconstituted with bacteriorhodopsin, forms a dicyclohexylcarbodiimide-inhibitable proton channel. Selective removal of the 116 kDa subunit transforms the proton ATPase from a Mg2+-activatable to a Ca2+-activatable ATPase. Subsequent dissociation and reconstitution of subunits reveals that the 70, 58, 40 and 33 kDa components are required, in composite, to form a functional ATP-hydrolytic core, and that no single subunit or subcomplex deficient in these subunits can catalyse ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1987

Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex.

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.

Animals↗
PubMed1987

Thermolabile proton translocating ATPase and pump activities in a clathrin-coated vesicle fraction from an acidification defective Chinese hamster cell line.

We have recently described a mutant of Chinese hamster ovary cells, termed G.7.1, that contains a temperature-sensitive, conditionally lethal mutation resulting in defective vacuolar acidification (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D.K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). To further characterize the lesion, clathrin-coated vesicles were partially purified from wild type and G.7.1 cells, and the thermolabilities of vanadate and oligomycin-insensitive, N-ethylmaleimide-sensitive, H+-ATPase activity, 32Pi-ATPase exchange activity, and proton pumping were compared. All three parameters of H+ pump activity were markedly diminished by preincubation at 44 degrees C for vesicles harvested from the G.7.1 cells, but not for those from wild type cells. Phosphatidylserine did not protect against heat inactivation in vesicle fractions prepared from G.7.1 cells. The results suggest that the mutation responsible for defective acidification in G.7.1 cells is expressed at the level of the proton pump of organelles present in our clathrin-coated vesicle-enriched preparation.

Adenosine Triphosphate↗
PubMed1987

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm.

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.

Adenosine Triphosphatases↗
PubMed1987

Natural history of borderline patients treated by intensive hospitalization.

The authors have the largest follow-up sample of long-term treatment ever done. They have studied almost 500 patients, 20 years after discharge from the long-term treatment unit at Psychiatric Institute. This article reviews some of their findings and identifies the patient population that requires and can benefit from long-term treatment.

Adult↗
PubMed1986

Isolation and reconstitution of the clathrin-coated vesicle proton translocating complex.

Clathrin-coated vesicles contain a proton translocating ATPase which is insensitive to azide but inhibited by N-ethylmaleimide. The ATP hydrolytic subunit of this proton pump has been solubilized, partially purified, and reconstituted into H+-ATPase-depleted coated vesicle membranes (Xie, X.-S., Stone, D.K., and Racker, E. (1984) J. Biol. Chem. 259, 11676-11678). In this communication we report that the entire proton transporting complex has been solubilized and purified 200-fold. The complex, when reconstituted into brain lipid liposomes, catalyzes azide-resistant, N-ethylmaleimide-sensitive H+ transport manifested as both generation of a pH gradient and an electrical gradient. The complex has an apparent molecular mass of 530 kDa.

Animals↗