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B P Crider

Publications and source records attributed to B P Crider.

At least 19 recordsLinked to original sources
PubMed2000

Identification and functional expression of four isoforms of ATPase II, the putative aminophospholipid translocase. Effect of isoform variation on the ATPase activity and phospholipid specificity.

ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg(2+)-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

Amino Acid Sequence↗
PubMed1999

Recombinant SFD isoforms activate vacuolar proton pumps.

The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V1) and an intramembranous proton channel, V0. V1 is comprised of 8-9 subunits including polypeptides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet). Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K. (1998) J. Biol. Chem. 273, 5878-5884). To determine the functional characteristics of the 57-kDa (SFDalpha)1 and 50-kDa (SFDbeta) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFDalpha and rSFDbeta, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of chromaffin granules has only the SFDalpha isoform in its native state and that rSFDalpha and rSFDbeta are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFDalpha and SFDbeta structurally interact not only with V1, but also withV0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.

Acridine Orange↗
PubMed1999

Identification and reconstitution of an isoform of the 116-kDa subunit of the vacuolar proton translocating ATPase.

We have identified a cDNA encoding an isoform of the 116-kDa subunit of the bovine vacuolar proton translocating ATPase. The predicted protein sequence of the new isoform, designated a2, consists of 854 amino acids with a calculated molecular mass of 98,010 Da; it has approximately 50% identity to the original isoform (a1) we described (Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D.K. (1994) J. Biol. Chem. 269, 17262-17266). Sequence comparison indicates that the a2 isoform is the bovine homologue of a 116-kDa polypeptide identified in mouse as an immune regulatory factor (Lee, C.-K., Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137-1144). The bovine a1 and a2 isoforms share strikingly similar structures with hydrophilic amino-terminal halves that are composed of more than 30% charged residues and hydrophobic carboxyl-terminal halves that contain 6-8 transmembrane regions. Northern blot analysis demonstrates that isoform a2 is highly expressed in lung, kidney, and spleen. To determine the possible role of the a2 isoform in vacuolar proton pump function, we purified from bovine lung a vacuolar pump proton channel (VO) containing isoform a2. This VO conducts bafilomycin-sensitive proton flow after reconstitution and acid activation, and supports proton pumping activity after assembly with the catalytic sector (V1) of vacuolar-type proton translocating ATPase (V-ATPase) and sub-58-kDa doublet, a 50-57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a2 isoform of the 116-kDa polypeptide functions as part of the proton channel of V-ATPases.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of the 50- and 57-kDa subunits of the bovine vacuolar proton pump.

The vacuolar type proton-translocating ATPase of clathrin-coated vesicles is composed of two large domains: an extramembranous catalytic sector and a transmembranous proton channel. In addition, two polypeptides of 50 and 57 kDa have been found to co-purify with the pump. These proteins, termed SFD (sub-fifty-eight-kDa dimer) activate ATPase activity of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Crider, B.P., Ma, Y.-M., and Stone, D. K. (1994) J. Biol. Chem. 269, 28509-25815). It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex responsible for the assembly of clathrin-coated pits, and that AP50 is essential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 31592-31597). We demonstrate through the use of anti-AP50 antibody, identical to that of the latter study, that hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional. To determine the true molecular identity of SFD, both the 50- and 57-kDa polypeptides were directly sequenced. A polymerase chain reaction-based strategy was used to screen a bovine brain cDNA library, yielding independent full-length clones (SFD-4A and SFD-21); these were identical in their open reading frames and encoded a protein with a predicted mass of 54,187 Da. The SFD-21 clone was then used in a reverse transcription-polymerase chain reaction-based strategy to isolate a related, but distinct, transcript present in bovine brain mRNA. The nucleotide and predicted amino acid sequences of this isolate are identical to SFD-21 except that the isolate contains a 54-base pair insert in the open reading frame, resulting in a protein with a predicted mass of 55,933 Da. Both clones had 16% identity to VMA13 of Saccharomyces cerevisiae. No sequence homology between the SFD clones and AP50 was detectable. Anti-peptide antibodies were generated against an epitope common to the two proteins and to the unique 18-amino acid insert of the larger protein. The former reacted with both components of native SFD, whereas the latter reacted only with the 57-kDa component. We term the 57- and 50-kDa polypeptides SFDalpha and SFDbeta, respectively.

Adaptor Protein Complex 2↗
PubMed1997

Subunit G of the vacuolar proton pump. Molecular characterization and functional expression.

The vacuolar type proton pump of clathrin-coated vesicles has a multisubunit ATP hydrolytic center that is peripheral to the membrane. Polypeptides present in this domain include the well characterized subunits A, B, C, D, E, and F; SFD, a dimer composed of 50- and 57-kDa polypeptides; and polypeptides termed G and H. Of these, subunits A, B, C, and E have been shown to be necessary but not sufficient for significant ATPase activity; in addition, either polypeptide G or H is also required for ATP hydrolysis (Xie, X.-S. (1996) J. Biol. Chem. 271, 30980-30985). In this study, the polypeptides G and H were purified and directly sequenced. Subsequent molecular analysis has revealed that these proteins are isoforms, which we designate G1 and G2. The cDNAs encoding the rat and bovine brain and chicken osteoclast forms of G1 have been cloned. The open reading frames of the rat and bovine clones encode hydrophilic proteins of 118 amino acids that differ at only five residues; bovine G1 has 36% identity with VMA10, a component of the proton channel of yeast. Northern blot analysis revealed a 1. 0-kilobase pair transcript encoding G1 in bovine brain, kidney, heart, and spleen. The cDNA encoding bovine polypeptide H was cloned and sequenced, revealing this protein to be 64% identical to G1, constituting isoform G2. In Northern blot analysis, a single 1. 7-kilobase pair transcript hybridized with a probe to G2 in brain, but not in heart, kidney, or spleen. An antibody against a bovine G1-specific domain reacts with V pump from bovine brain, kidney, and chromaffin granule, whereas an anti-G2 antibody reacts only with proton pump from brain. The bovine forms of G1 and G2 were subsequently expressed in Escherichia coli and Sf9 cells, respectively, and purified to homogeneity. Reconstitution of ATP hydrolysis was achieved by combination of recombinant subunits A, B, C, and E with either recombinant G1 or G2, demonstrating the role of these isoforms in pump function.

Adenosine Triphosphate↗
PubMed1996

Identification of a 14-kDa subunit associated with the catalytic sector of clathrin-coated vesicle H+-ATPase.

The clathrin-coated vesicle H+-ATPase is composed of a peripheral catalytic sector (VC) and an integral membrane proton channel (VB), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13, 370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated VC. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue.

Amino Acid Sequence↗
PubMed1994

Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase.

Vacuolar-type proton pumps are complex heterooligomers. When dissociated into subcomplexes and subunits, the partial reactions of ATP hydrolysis and transmembranous proton flow can be assigned to isolated domains. Data suggest that the molecular site of ATP hydrolysis resides within the 70-kDa subunit but that ATPase activity likely requires at least three additional subunits of 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now cloned and sequenced the 70-kDa subunit from bovine brain and have expressed the protein in insect Sf9 (Spodoptera frugiperda) cells with a recombinant baculovirus. When purified, the protein has no significant ATPase activity but can be photoaffinity labeled with [alpha 32P]ATP and UV irradiation with an apparent Kd of 35 microM. When reconstituted with biochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombinant 70-kDa subunit restores Ca(2+)-activated ATP hydrolysis to a specific activity of 0.6 mumol P(i).mg protein-1.min-1, thus demonstrating that ATP hydrolysis in vacuolar-type proton pumps is dependent upon both the 70-kDa subunit as well as multi-subunit interactions.

Adenosine Triphosphate↗
PubMed1994

Role of a 50-57-kDa polypeptide heterodimer in the function of the clathrin-coated vesicle proton pump.

The vacuolar-type proton-translocating ATPase of clathrin-coated vesicles is composed of an integral membrane proton channel (VB) and a peripheral catalytic sector (VC). Native enzyme can catalyze the hydrolysis of both MgATP and CaATP and support proton pumping when reconstituted into liposomes. In contrast, isolated VC catalyzes only Ca(2+)-activated ATP hydrolysis and cannot support proton pumping when reconstituted into liposomes (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We now report that solubilized isolated VC can be reassembled with purified VB to restore properties of native enzyme, including Mg(2+)-activated ATP hydrolysis and proton-pumping capability. Investigation of this reassembly revealed that a heterodimer, composed of polypeptides of 50 and 57 kDa, stimulates Ca(2+)-activated ATPase activity of isolated VC 2-fold and Mg(2+)-activated ATPase activity catalyzed by the reassembled pump 9-fold. Moreover, this heterodimer stimulated proton transport by the reassembled pump > 20-fold. When separated from the proton pump, the dimer has no detectable kinase activity. Maximal stimulation occurs at a molar ratio of heterodimer to reassembled pump of 3, implying a structural, nonenzymatic mechanism. These data indicate that the 50-kDa and/or the 57-kDa polypeptide likely plays an essential and potentially regulatory role in the function of the proton-translocating ATPase of clathrin-coated vesicles.

Adenosine Triphosphate↗
PubMed1994

Bafilomycin inhibits proton flow through the H+ channel of vacuolar proton pumps.

Vacuolar-type proton-translocating ATPases are complex heterooligomers that are characterized by a specific inhibition by bafilomycin A1. These enzymes have a peripheral ATP hydrolytic domain as well as a transmembranous sector. The transmembranous sector has been isolated by glycerol gradient centrifugation, and this subcomplex is composed of polypeptides of 116, 39, and 17 kDa. Both this sector and native holoenzyme were reconstituted into potassium-loaded (150 mM KCl) liposomes prepared from pure lipids. When diluted into potassium-free buffer, a valinomycin-induced membrane potential did not drive proton uptake, as assessed by acridine orange quenching. In contrast, pretreatment of both the reconstituted proton pump and isolated transmembranous sector at pH 4.2 activated a latent proton conductance. Bafilomycin A1 (1 nM) inhibited ATP-energized proton pumping catalyzed by the proton pump, as well as membrane potential-driven proton flow through both the acid-activated proton pump and the isolated proton pore. Thus bafilomycin A1 inhibits vacuolar proton pumps by blocking proton conduction through the proton pore, which we term VB.

Animals↗
PubMed1994

Alternative mRNA splicing generates tissue-specific isoforms of 116-kDa polypeptide of vacuolar proton pump.

The cDNA encoding the 116-kDa polypeptide of the bovine brain vacuolar-type proton translocating ATPases has been cloned and sequenced. One of five clones differed from all others in that it contained an 18-base pair deletion within the coding region, whereas it was identical to the other clones in overlapping coding and noncoding regions, indicating that this heterogeneity arises through an alternative splicing mechanism. By conventional Northern analysis, only one 4.1-kilobase mRNA was identified in bovine brain, heart, kidney, liver, and spleen. However, a polymerase chain reaction-based analysis revealed two species of mRNA with a tissue-specific distribution. Type I, containing the 18-base pair insert, was found in brain, whereas the truncated (Type II) form was found in all tissues examined. Similar tissue distributions of rat mRNA were observed. The deletion site accounting for this variability occurs within a predicted protease sensitivity motif (PEST site), suggesting that differences in the biological half-life of the two 116-kDa isoforms may exist.

Alternative Splicing↗
PubMed1993

Isolation of a protein activator of the clathrin-coated vesicle proton pump.

An activator of the clathrin-coated vesicle proton translocating ATPase has been purified 1600-fold from bovine brain. The activator, which requires detergent (polyoxyethylene 9-lauryl ether) for release from clathrin-coated vesicles, is heat-stable, trypsin-sensitive, and has an apparent molecular mass of about 6 kDa as determined by high performance liquid chromatography. The activator stimulates the purified H(+)-ATPase of coated vesicles over 50-fold under acidic conditions. Similarly, the activator stimulates proton pumping catalyzed by the reconstituted proton pump. Importantly, this stimulation of proton pumping is observed only when the activator is reconstituted into the interior of the proteoliposomes. Moreover, the activator protein is demonstrated to protect, and co-sediment with, purified proton pump during glycerol gradient centrifugation performed in the presence of ATP. These observations support the notion that this activator serves to determine the pH set point of acidic endomembranes through interactions with the transmembranous sectors of the proton pump.

Animals↗
PubMed1990

Inhibition of glucose transport into rat islet cells by immunoglobulins from patients with new-onset insulin-dependent diabetes mellitus.

Because glucose-stimulated insulin secretion is selectively impaired during the development of insulin-dependent diabetes mellitus (IDDM), we tested the possibility that the glucose transporter of pancreatic islet beta cells is a target of the autoimmune process in patients with IDDM. We measured the uptake of 3-O-methyl-beta-D-glucose by dispersed islet cells from rats after a 15-minute incubation with purified IgG from 27 patients with newly diagnosed IDDM, 28 normal subjects, and 5 patients with non-insulin-dependent diabetes mellitus (NIDDM). The IgG fractions from 26 of the 27 patients with IDDM (96 percent), but from none of the 5 patients with NIDDM, reduced the initial rates of 3-O-methyl-beta-D-glucose uptake to at least 1 SD below the mean of the rates observed in the presence of IgG fractions from normal subjects (P less than 0.001). In contrast, the uptake of L-leucine by islet cells was not affected by any of the IgG fractions. The inhibitory activity of IgG from the patients with IDDM was abolished by preincubation with islet cells and membranes from hepatocytes, which contain the same glucose transporter as beta cells, but not with erythrocytes, which do not contain this transporter. We conclude that IgG from patients with IDDM of recent onset, but not from those with NIDDM, inhibits glucose uptake by rat islet cells. The results are consistent with the presence of an antibody against a protein involved in glucose transport by beta cells that would thereby impair glucose-stimulated insulin secretion.

3-O-Methylglucose↗
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

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

Increases in Na+,K+-ATPase activity of erythrocytes and skeletal muscle after chronic ethanol consumption: evidence for reduced efficiency of the enzyme.

Increased Na+,K+-ATPase activity observed after chronic ethanol consumption has been examined to determine whether the increase is due to changes in the kinetic properties of the enzyme or increases in the amount of enzyme in the membranes examined. In skeletal muscle and erythrocyte ghosts from rat, as well as from humans, increased Na+,K+-ATPase activity in ethanol-consuming individuals was not accompanied by an increase in the number of ouabain binding sites. In studies with intact human erythrocytes, similar ouabain-sensitive 22Na+ and 86Rb+ pumping rates were observed between normal and ethanol-consuming individuals and the Na+ to Rb+ pumping ratio was found to be 1.5 in all cases. However, ouabain-sensitive lactate plus Pi formation was increased in cells from alcoholic individuals. Thus these data suggest that increased enzyme activity may be due to a kinetic alteration of the Na+,K+-ATPase and that the enzyme may be less efficient in coupling ion pumping to ATP hydrolysis than the enzyme in normal cells.

Adult↗