Biomedical subjects

J Zhou

Publications and source records attributed to J Zhou.

At least 595 records · Page 33Linked to original sources

PubMed1997

A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer.

The ETS family of genes are implicated in cancers such as Ewings sarcoma, acute myeloid leukemia and chronic myelomonocytic leukemia. Further, they have important functions in embryonic development. Hence, identification and characterization of members of this family are important. We identify a novel ETS family member, ELF3, and report its human and murine cDNA sequences. The mouse cDNA has an alternatively spliced transcript with an extra 60 bp inserted. Hence we present the organization of the murine Elf3 gene together with its exon/intron structure. This gene consists of 9 exons and 8 introns spanning 4.8 kb. ELF3 binds and transactivates ETS sequences and interestingly also shows the ability to bind a GGAT-like purine core, a preferential ETS1/ETS2 type binding site. The expression of ELF3, unlike most other ETS family members, is absent in hematopoietic cells and hematopoietic organs in humans and mice. Intriguingly, the gene is specifically expressed in cell lines of epithelial origin and in organs such as lung, stomach, intestine, kidney that have specialized epithelial cells. We localize the human gene to 1q32.2, a region that is amplified in epithelial tumors of the breast, lung and prostate. Finally, we show that ELF3 expression is increased in a lung carcinoma and adenocarcinoma, as compared to normal tissue. ELF3 is also expressed in cell lines derived from lung cancers. These results suggest that this novel ETS gene may be involved in lung tumorigenesis.

Amino Acid Sequence↗

PubMed1997

Left ventricular diastolic filling with an implantable ventricular assist device: beat to beat variability with overall improvement.

OBJECTIVES: We studied the effects of left ventricular (LV) unloading by an implantable ventricular assist device on LV diastolic filling. BACKGROUND: Although many investigators have reported reliable systemic and peripheral circulatory support with implantable LV assist devices, little is known about their effect on cardiac performance. METHODS: Peak velocities of early diastolic filling, late diastolic filling, late to early filling ratio, deceleration time of early filling, diastolic filling period and atrial filling fraction were measured by intraoperative transesophageal Doppler echocardiography before and after insertion of an LV assist device in eight patients. A numerical model was developed to simulate this situation. RESULTS: Before device insertion, all patients showed either a restrictive or a monophasic transmitral flow pattern. After device insertion, transmitral flow showed rapid beat to beat variation in each patient, from abnormal relaxation to restrictive patterns. However, when the average values obtained from 10 consecutive beats were considered, overall filling was significantly normalized from baseline, with early filling velocity falling from 87 +/- 31 to 64 +/- 26 cm/s (p < 0.01) and late filling velocity rising from 8 +/- 11 to 32 +/- 23 cm/s (p < 0.05), resulting in an increase in the late to early filling ratio from 0.13 +/- 0.18 to 0.59 +/- 0.38 (p < 0.01) and a rise in the atrial filling fraction from 8 +/- 10% to 26 +/- 17% (p < 0.01). The deceleration time (from 112 +/- 40 to 160 +/- 44 ms, p < 0.05) and the filling period corrected by the RR interval (from 39 +/- 8% to 54 +/- 10%, p < 0.005) were also significantly prolonged. In the computer model, asynchronous LV assistance produced significant beat to beat variation in filling indexes, but overall a normalization of deceleration time as well as other variables. CONCLUSIONS: With LV assistance, transmitral flow showed rapidly varying patterns beat by beat in each patient, but overall diastolic filling tended to normalize with an increase of atrial contribution to the filling. Because of the variable nature of the transmitral flow pattern with the assist device, the timing of the device cycle must be considered when inferring diastolic function from transmitral flow pattern.

Adult↗

PubMed1997

Residues of the cytoplasmic domain of MotA essential for torque generation in the bacterial flagellar motor.

The MotA protein of Escherichia coli is a component of the flagellum that functions, together with MotB, in transmembrane proton conduction. MotA and MotB are believed to form the stator of the flagellar motor. They are integral membrane proteins; MotA has a large (ca 22 kDa) domain in the cytoplasm, and MotB a much smaller one (ca 3 kDa). Recent work suggests that cytoplasmically located parts of MotA and/or MotB might be present at the active site for torque generation in the motor. To test the proposal that the cytoplasmic domain of MotA functions in torque generation, and to identify the amino acid residues most important for function, we have carried out a mutational analysis of this domain. Using random mutagenesis, many mutations of cytoplasmic residues of MotA were isolated, which either abolish or impair torque generation. In most cases the residues affected are not conserved, and many of the replacements involve loss or gain of a proline residue, which suggests that these mutations disrupt function by altering the protein conformation rather than by directly affecting residues of an active site. Using site-directed mutagenesis, the conserved residues in the cytoplasmic domain of MotA were replaced, either singly or, in the case of charged residues, in various combinations. The results identify four residues of MotA that are important for motor function. These are Arg90 and Glu98, located in the cytoplasmic domain, and Pro173 and Pro222, located at the interface between the cytoplasmic domain and the membrane-spanning domain. Possible roles for these residues in torque generation are discussed.

Amino Acid Sequence↗

PubMed1997

Attenuation of cerebral vasospasm by systemic administration of an endothelin-A receptor antagonist, TBC 11251, in a rabbit model of subarachnoid hemorrhage.

Cerebral vasospasm is a major complication of subarachnoid hemorrhage (SAH) after the rupture of an intracranial aneurysm. Although the cause of cerebral vasospasm has not been fully established, several lines of evidence suggest that the vasoconstrictor peptide endothelin (ET) may play a crucial role. In the present study the potential of TBC 11251 (TBC), a newly developed ET(A) receptor antagonist, to prevent and/or reverse cerebral vasospasm was examined in a well-established rabbit model of SAH. Sixty-five New Zealand White rabbits were assigned to one of six groups. Experimental SAH was induced in rabbits comprising five of the groups by injecting autologous arterial blood into the cisterna magna. The treatment groups were as follows: 1) control (no SAH); 2) SAH only; 3) SAH + placebo at 24 and 36 hours (24/36); 4) SAH + TBC (24/36); 5) SAH + placebo twice daily (BID); and 6) SAH + TBC BID. All drug-treated animals received an intravenous dosage of 5 mg/kg TBC. After 48 hours, the animals were killed by intracardiac perfusion with fixative. The brainstems were removed and the basilar arteries (BAs) were prepared for histological examination. The cross-sectional area of each BA was measured using computer-assisted videomicroscopy by an investigator blind to the group from which it came. A one-way analysis of variance and paired group mean comparisons with the post-hoc Fisher least significant difference test were used for analysis of BA diameters and physiological parameters. The model provided reliable vasospasm, with the mean BA cross-sectional area constricting from 0.388 mm2 in the control group to 0.106 mm2 (27.4% of control) in the SAH only group. Treatment with TBC (24/36) after SAH (reversal protocol) produced a mean BA area of 0.175 mm2 (44.2% of control) which, although larger than the placebo group value of 0.135 mm2 (39.9% of control), was not statistically significant. However, treatment with TBC BID (prevention protocol) produced a mean BA area of 0.303 mm2 (78.1% of control) compared with the placebo BID value of 0.134 mm2 (34.6% of control); this effect was statistically significant (p < 0.01). There were no side effects noted and no differences in the mean arterial pressures between drug and placebo groups. These findings demonstrate that systemic administration of the ET(A) receptor antagonist TBC significantly attenuates cerebral vasospasm after SAH when given as a preventative therapy, and they provide additional support for the role of ET in the establishment of vasospasm.

Journal Article↗

PubMed1997

SNAP-25 is phosphorylated by glucose and GLP-1 in RIN 1046-38 cells.

We investigated the possibility that tyrosine phosphorylation might play a role in insulin secretion in the insulinoma cell line, RIN 1046-38 cells. At least 4 proteins of 18, 25, 35, and 46 kDa size were found to be tyrosine phosphorylated in the presence of glucose and an insulin secretagogue, glucagon-like peptide-1 (GLP-1). The addition of glucose and GLP-1 to cells that were exposed to the tyrosine kinase inhibitor genistein resulted in a decrease in the extent of phosphorylation of the 18, 25, and 35 kDa proteins and a concomitant reduction in insulin secretion, whereas treatment with vanadate, a tyrosine phosphatase inhibitor, led to enhanced responses. Immunoprecipitation of cellular proteins with an anti-phosphotyrosine antibody followed by immunoblotting with a specific monoclonal antibody to SNAP-25 (synaptosome-associated protein of 25 kDa) revealed that the 25 kDa protein is SNAP-25. These results suggest that tyrosine phosphorylation of SNAP-25 may be involved in the regulation of insulin secretion in RIN 1046-38 cells.

Animals↗

PubMed1997

A kinetic model with ordered cytoplasmic dissociation for SUC1, an Arabidopsis H+/sucrose cotransporter expressed in Xenopus oocytes.

To elucidate the kinetic properties of the Arabidopsis H+/sucrose cotransporter, SUC1, with respect to transmembrane voltage and ligand concentrations, the transport system was heterologously expressed in Xenopus laevis oocytes. Steady-state plasma membrane currents associated with transport of sucrose were measured with two-electrode voltage clamp over the voltage range -180 to +40 mV as a function of extracellular pH and sugar concentrations. At any given voltage, currents exhibited hyperbolic kinetics with respect to extracellular H+ and sugar concentrations, and this enabled determination of values for the maximum currents in the presence of each ligand (iHmax, iSmax for H+ and sucrose) and of the ligand concentrations eliciting half-maximal currents (KHm, KSm). The iHmax and iSmax exhibited marked and statistically significant increases as a function of increasingly negative membrane potential. However, the KHm and KSm decreased with increasingly negative membrane potential. Furthermore, at any given voltage, iSmax increased and KSm decreased as a function of the external H+ concentration. Eight six-state carrier models-which comprised the four possible permutations of intracellular and extracellular ligand binding order, each with charge translocation on the sugar-loaded or -unloaded forms of the carrier-were analyzed algebraically with respect to their competence to account for the ensemble of kinetic observations. Of these, two models (first-on, first-off and last-on, first-off with respect to sucrose binding as it passes from outside to inside the cell and with charge translocation on the loaded form of the carrier) exhibit sufficient kinetic flexibility to describe the observations. Combining these two, a single model emerges in which the binding on the external side can be random, but it can only be ordered on the inside, with the sugar dissociating before the proton.

Animals↗

PubMed1997

Steroid receptor coactivator-1 is a histone acetyltransferase.

Steroid receptors and coactivator proteins are thought to stimulate gene expression by facilitating the assembly of basal transcription factors into a stable preinitiation complex. What is not clear, however, is how these transcription factors gain access to transcriptionally repressed chromatin to modulate the transactivation of specific gene networks in vivo. The available evidence indicates that acetylation of chromatin in vivo is coupled to transcription and that specific histone acetyltransferases (HATs) target histones bound to DNA and overcome the inhibitory effect of chromatin on gene expression. The steroid-receptor coactivator SRC-1 is a coactivator for many members of the steroid-hormone receptor superfamily of ligand-inducible transcription factors. Here we show that SRC-1 possesses intrinsic histone acetyltransferase activity and that it also interacts with another HAT, p300/CBP-associated factor (PCAF). The HAT activity of SRC-1 maps to its carboxy-terminal region and is primarily specific for histones H3 and H4. Acetylation by SRC-1 and PCAF of histones bound at specific promoters may result from ligand binding to steroid receptors and could be a mechanism by which the activation functions of steroid receptors and associated coactivators enhance formation of a stable preinitiation complex, thereby increasing transcription of specific genes from transcriptionally repressed chromatin templates.

Acetylation↗

PubMed1997

Chimeric papillomavirus-like particles.

We have constructed chimeric papillomavirus-like particles (CVLPs) by replacing the 34-carboxy-terminal amino acids of the HPV 16 L1 protein with various parts of the HPV 16 E7 protein. Chimeric proteins were expressed by recombinant baculoviruses and analyzed by electron microscopy for their ability to assemble into virus capsids. We were able to produce CVLPs in high efficiencies with inserts of up to 60 amino acids. CVLPs are able to induce a neutralizing antibody response, assayed by inhibition of hemagglutination of mouse erythrocytes. CVLPs are interacting with the putative receptor for papillomaviruses as they were shown to hemagglutinate mouse red blood cells and bind to and penetrate cells in vitro. As CVLPs follow a similar intracellular pathway as observed earlier for BPV VLPs, we speculate that CVLPs can be used to deliver peptides into mammalian cells in vitro and in vivo, possibly reaching the pathway for MHC class I presentation.

Amino Acid Sequence↗

PubMed1997

Seminiferous tubule basement membrane. Composition and organization of type IV collagen chains, and the linkage of alpha3(IV) and alpha5(IV) chains.

Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six alpha-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of alpha3(IV) chain (40%) and the alpha4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six alpha(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of alpha(IV) chains, were characterized for their alpha(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with alpha(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the alpha1(IV) and alpha2(IV) chains and two novel networks, one composed of the alpha1(IV)-alpha6(IV) chains and the other composed of the alpha3(IV)-alpha6(IV) chains. The results establish a structural linkage between the alpha3(IV) and alpha5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the alpha5(IV) chain cause defective assembly of the alpha3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.

Animals↗

PubMed1997

Glucocorticoid regulation of natural cytotoxicity: effects of cortisol on the phenotype and function of a cloned human natural killer cell line.

The ability of glucocorticoids to suppress cellular immune functions, including the cytotoxic activity of natural killer cells, is well known. However, the molecular mechanism(s) of glucocorticoid-mediated suppression of cellular cytotoxicity mediated by natural killer cells is not understood. We have investigated the effects of cortisol on protein expression and cytotoxic function of natural killer cells using NK3.3, a well-characterized, cloned human natural killer cell line. Cortisol, at concentrations up to 2 microM, does not significantly alter the viability or proliferative capacity of NK3.3 cells. However, micromolar concentrations of cortisol induce the expression of a small set of proteins which are not synthesized by NK3.3 cells in the absence of cortisol, and repress the synthesis of another set of proteins including several phenotypic determinants and cytokines. In the presence of added cortisol, the synthesis of perforin mRNA was partially repressed. However, the most striking effect of cortisol on this NK clone was its repression of granzyme A synthesis. In conjunction with the downregulation of adhesion proteins, NK3.3 cells cultured in the presence of cortisol exhibit a reduced capacity to form conjugates with K562 target cells. Whereas cortisol treatment of NK3.3 cells causes an approximately 50% decrease in their ability to form conjugates with K.562 target cells, the cytotoxic function of these cells is completely abolished under the same conditions. This first report of hormonal regulation of granzyme expression and the strong correlation between granzyme A repression and cytotoxic function suggests that cortisol may regulate NK function by repression of granzyme A synthesis. In addition to demonstrating the significant influence of cortisol on natural killer cell function, these studies provide a model system for elucidation of molecular mechanism(s) whereby glucocorticoids repress cellular immune function, especially with respect to natural killer cells.

Anti-Inflammatory Agents↗

PubMed1997

The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes.

In tomato, the Pto kinase confers resistance to bacterial speck disease by recognizing the expression of a corresponding avirulence gene, avrPto, in the pathogen Pseudomonas syringae pv. tomato. Using the yeast two-hybrid system, we have identified three genes, Pti4, Pti5 and Pti6, that encode proteins that physically interact with the Pto kinase. Pti4/5/6 each encode a protein with characteristics that are typical of transcription factors and are similar to the tobacco ethylene-responsive element-binding proteins (EREBPs). Using a gel mobility-shift assay, we demonstrate that, similarly to EREBPs, Pti4/5/6 specifically recognize and bind to a DNA sequence that is present in the promoter region of a large number of genes encoding 'pathogenesis-related' (PR) proteins. Expression of several PR genes and a tobacco EREBP gene is specifically enhanced upon Pto-avrPto recognition in tobacco. These observations establish a direct connection between a disease resistance gene and the specific activation of plant defense genes.

Amino Acid Sequence↗

PubMed1997

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed delta-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with beta-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Amino Acid Sequence↗

PubMed1997

Influence of BDNF on the expression of the dopaminergic phenotype of tissue used for brain transplants.

Brain-derived neurotrophic factor (BDNF) has previously been shown by this laboratory among others to promote survival and differentiation of central dopaminergic neurons and to stimulate expression of the dopaminergic phenotype in fetal cerebrocortex in vitro. We have examined the effect of BDNF antibody on nigral dopaminergic neurons in vivo and in vitro. It reduced the survival of rat fetal dopaminergic neurons in culture (up to 40% died). The BDNF antibody also caused ipsilateral rotation after a single in vivo intranigral injection in the adult rats. Pre-treatment of fetal nigral neurons with BDNF improved the performance of dopaminergic cells in fetal nigral transplants based on surviving TH+ cells numbers. Thus, parkinsonian rats receiving fetal nigral cells treated with BDNF showed a significantly greater reduction of turning over the 3 weeks following transplantation, compared with the rats receiving untreated nigral transplants. However, the average number of tyrosine hydroxylase (TH)-positive neurons in the grafts of rats receiving fetal nigral cells treated with BDNF was 211 +/- 35 which was only about 20% of the cell number (1012 +/- 223, mean +/- S.E.M.) found in those receiving untreated nigral transplants. These results suggest that pretreatment of nigral dopaminergic neurons with BDNF may improve their functional performance, but not their survival in transplants. The ability of artificially induced cerebrocortical 'dopaminergic' cells to ameliorate behavioral asymmetry of Parkinsonian rats was assessed. A proportion (1.0% maximum) of the TH+ neurons in these transplants survived in the host brain and were likely to be responsible for the prominent reduction in rotation scores observed to occur 6 weeks after implantation. Thus, the combined treatment of fetal cerebral cortex with BDNF and dopamine created long-lived TH-expressing neuronal populations which were very effective in alleviating the rat parkinsonian model, and thus may be suitable for use in transplantation in treating human Parkinson's disease.

Animals↗

PubMed1997

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed gamma-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with gamma-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Disheveled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Amino Acid Sequence↗

PubMed1997

Binding of thyroxine analogs to human liver aldehyde dehydrogenases.

A fragment of a cytosolic thyroid-hormone-binding protein from Xenopus liver was reported to be 92-100% identical to residues 236-258 in several cytosolic aldehyde dehydrogenases [Yamauchi, K. & Tata, J. R. (1994) Eur. J. Biochem. 225, 1105-1112], which have been proposed to form part of the hinge region necessary to bind the adenosine moiety of NAD. Here we investigated the effects of two thyroxine analogs, 3,3',5-triiodo-L-thyronine and 3,3',5-triiodothyroacetic acid, on purified human liver mitochondrial and cytosolic aldehyde dehydrogenases. The compounds were found to be competitive inhibitors against NAD and uncompetitive inhibitors with respect to aldehyde. At pH 7.4, the apparent Ki values were in the micromolar range when the concentration of NAD was varied. The inhibition against recombinantly expressed mutant forms of aldehyde dehydrogenase, which possessed diminished NAD binding, was determined. Essentially no differences were found between the native enzyme and the mutants, showing that the analog binding was not affected by altering the NAD-binding site. Furthermore, the analogs could displace NAD but not NADH from the enzyme. These findings indicated that the binding of NAD differed from that of NADH, and that aldehyde dehydrogenases, like other dehydrogenases, can be inhibited by thyroxine analogs.

Acetophenones↗

PubMed1997

Feedback inhibition of Ca2+ channels by Ca2+ depends on a short sequence of the C terminus that does not include the Ca2+ -binding function of a motif with similarity to Ca2+ -binding domains.

alpha(1C)- and alpha(1E)-based Ca2+ channels differ in that the former are inhibited by Ca2+ entering through its pore, while the latter are not. It has been proposed on the basis of analysis of alpha(1E)/alpha(1C) chimeras that the molecular determinants responsible for Ca2+ inhibition involve both a conserved Ca2+-binding motif (EF hand) plus additional sequences located C-terminal to the EF hand. Through construction of similar alpha(1E)/alpha(1C) chimeras, we transferred Ca2+ inhibition from alpha(1C) to alpha(1E) by replacing a 134-aa segment of alpha(1E) with the homologous 142-aa segment of alpha(1C). This segment is located immediately after the proposed Ca2+ -binding EF hand motif. Replacement of the alpha(1C) EF hand with the corresponding EF hand of alpha(1E) did not interfere with inhibition of alpha(1C) by Ca2+, and a triple mutant of alpha(1C), alpha(1C)[D1535A,E1537A,D1546A], that disrupts the potential Ca2+-coordinating ability of the EF hand continued to be inhibited by Ca2+. These results indicate that a small portion of the alpha(1C) C terminus is essential for inhibition by Ca2+ and place the Ca2+ -binding site anywhere in alpha(1C), with the exception of its EF hand-like motif.

Amino Acid Sequence↗

PubMed1997

Is there a catalytic base in the active site of cAMP-dependent protein kinase?

The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.

Adenosine Triphosphate↗

PubMed1997

A Xenopus oocyte beta subunit: evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit.

Two closely related beta subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus beta antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca2+ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian alpha1 cRNAs (alpha(1C) and alpha(1E)); (ii) coinjection of a Xenopus beta antisense oligonucleotide and excess rat beta cRNA rescued expression of alpha1 Ca2+ channel currents; and (iii) coinjection of mammalian alpha1 cRNA with cRNA encoding either of the two Xenopus beta subunits facilitated both activation and inactivation of Ca2+ channel currents by voltage, as happens with most mammalian beta subunits. The Xenopus beta subunit cDNAs (beta3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 beta subunit. We propose that "alpha1 alone" channels are in fact tightly associated alpha1beta3xo channels, and that effects of exogenous beta subunits are due to formation of higher-order [alpha1beta]beta(n) complexes with an unknown contribution of beta3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition alpha1beta, are [alpha1beta]beta(n) complexes that associate with alpha2delta and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of alpha(1E) have a beta subunit binding domain. This beta binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel alpha1 subunits.

Amino Acid Sequence↗