cDNA cloning of possible mammalian homologs of the yeast secretory pathway Ca(2+)-transporting ATPase.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
D M Clarke
Publications and source records attributed to D M Clarke.
Molecular cloning and tissue distribution of alternatively spliced mRNAs encoding possible mammalian homologues of the yeast secretory pathway calcium pump.
Rat stomach and testis cDNAs corresponding to two alternatively spliced mRNAs encoding variants of a P-type ion-transport ATPase that closely resembles the yeast secretory pathway Ca2+ pump have been isolated and characterized. A partial kidney cDNA was identified previously using an oligonucleotide probe corresponding to part of the sarcoplasmic reticulum Ca(2+)-ATPase [Gunteski-Hamblin, A., Greeb, J., & Shull, G.E. (1988) J. Biol. Chem. 263, 15032-15040]. In the present study, we first isolated and characterized a stomach cDNA that contains the entire coding sequence. The 919 amino acid enzyme has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. Northern blot analyses demonstrate that 3.9- and 5-kilobase mRNAs corresponding to the cDNA were present in all tissues examined, suggesting that the protein it encodes performs a housekeeping function. Rat testis also contained a 3.7-kilobase mRNA that hybridized with a probe from the 5' end of the stomach cDNA but did not hybridize with a probe from the 3' end. Cloning and characterization of cDNAs corresponding to the smaller testis mRNA revealed that it is derived from the same gene but encodes a variant of the enzyme in which the C-terminal residue, Val-919, is replaced by the sequence Phe-919-Tyr-Pro-Lys-Ile-923. Similarity comparisons show that the two enzymes are more closely related to the known Ca2+ pumps than to other P-type ATPases.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Necrotizing enterocolitis in a patient with AIDS.
Explore the source record for details and available documents.
Acquired Immunodeficiency Syndrome↗
Site-directed mutagenesis of the Ca2+ ATPase of sarcoplasmic reticulum.
We have cloned cDNA encoding the Ca2+ ATPase from fast-twitch skeletal muscle and, on the basis of its amino acid sequence and immunological studies of its topology, have made deductions concerning its secondary structure and active sites. These deductions have led us to test models for Ca2+ transport through expression of the protein in functional form in COS-1 cells, mutagenesis, and measurement of altered function. Mutation of about 250 of the 1000 amino acids making up the Ca2+ pump has indicated that the sites of high affinity Ca2+ binding are located in the center of the transmembrane domain and are made up from residues located in transmembrane sequences M4, M5, M6 and M8. The ATP binding site appears to be located in the headpiece and is made up from a series of loop sequences connecting alternating alpha helices and beta strands. Sites of conformational interaction appear in all domains throughout the Ca2+ pump. In our present model, Ca2+ transport occurs through binding to high affinity sites accessible to the cytoplasm in the E1 conformation, followed by release to the lumen from low affinity sites which form during the ATP-induced transition of the protein from the E1 to the E2 conformation.
Animals↗
The prevalence of psychiatric morbidity in general hospital inpatients.
The aim of this study was to examine the prevalence of psychiatric morbidity in a sample of medical and surgical inpatients in an Australian general hospital. Using the 60-item General Health Questionnaire, the estimated prevalence was 30% (previous studies have yielded estimates generally between 20 and 50%). The prevalence of morbidity was significantly higher in medical (45%) than in surgical (23%) inpatients. Twelve percent of patients (20% of medical patients and 8% of surgical patients) satisfied DSM-III criteria for a current Major Depressive Episode. Anxiety scores on the State-Trait Anxiety Inventory were higher than those reported in general population samples. There were no significant differences between males and females on any scores. The problems associated with the definition and identification of depressive and anxiety syndromes in medical and surgical inpatients are discussed, whilst the importance of this task is emphasized.
Anxiety Disorders↗
Illness behaviour as a determinant of referral to a psychiatric consultation/liaison service.
Recognition of psychiatric disturbance and rate of referral to psychiatric units in general hospitals falls far short of the prevalence of psychiatric morbidity in such hospitals. This study has sought to determine whether particular patterns of "illness behaviour" contribute to the decision to refer. A group of patients referred to a psychiatric service was compared to a group of non-referred patients using the General Health Questionnaire (GHQ) as a general measure of morbidity, the Inventory to Diagnose Depression (IDD), the State-Trait Anxiety Inventory (STAI) and the Illness Behaviour Questionnaire (IBQ). The rate of referral to the service was 2.9%. The estimated prevalence of psychiatric morbidity was 30%. Referred patients scored more highly on all measures of morbidity (GHQ, IDD, STAI) and on the IBQ scale Affective Disturbance, and scored lower on the Denial Scale. Data are reported which suggest that patients with a strong somatic focus are less likely to be referred for psychiatric assessment despite the presence of significant psychiatric morbidity. The implications for health care delivery and identification of psychiatric morbidity are discussed.
Adult↗
Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.
Amino acids in three highly conserved segments of the Ca2(+)-ATPase. Asp-Pro-Pro-Arg604, Thr-Gly-Asp627, Thr-Gly-Asp703 as well as Asp707, have been proposed to participate in formation of the nucleotide binding site. We have tested this hypothesis by investigating the properties of mutants with alterations to amino acids within these segments. Most of the mutants were found to be defective in Ca2+ transport function. The inactive mutants could be separated into two classes on the basis of the kinetics of phosphoenzyme intermediate formation and decomposition. One group, Asp601----Asn, Pro603----Leu, Asp627----Glu, and Asp703----Asn, formed phosphoenzyme intermediates with ATP in the presence of Ca2+ and with inorganic phosphate only in the absence of Ca2+, indicating that both the high affinity Ca2+ binding sites and the nucleotide binding sites were intact. In each of these mutants, however, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive phosphoenzyme intermediate very slowly, relative to the wild-type enzyme. Thus the inability of these mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. Another group, Asp601----Glu, Pro603----Gly, Asp707----Glu, and Asp707----Asn, did not form detectable phosphoenzyme intermediates from either ATP or Pi. Although we have demonstrated an effect on Ca2+ transport of mutations in each of the highly conserved regions predicted to be involved in ATP binding, we cannot yet define their roles in ATP-dependent Ca2+ transport.
Adenosine Triphosphate↗
The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the luminal surface of the Ca2(+)-ATPase of sarcoplasmic reticulum.
The epitope for monoclonal antibody A20 was mapped to amino acids 870-890 of the Ca2(+)-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The antibody did not react with the epitope in intact sarcoplasmic reticulum vesicles but reacted with the epitope when the vesicles were solubilized with the detergent C12E8 or made permeable by incubation in a hypotonic medium. By contrast, antibody A52, which binds to a cytoplasmic epitope consisting of amino acids 657-672, reacted with the Ca2(+)-ATPase in vesicular, permeabilized vesicular, and C12E8-solubilized states. These results clearly demonstrate that antibody A20 binds to a luminal epitope and provide the first demonstration that a specific segment of the Ca2(+)-ATPase is located on the luminal surface of the sarcoplasmic reticulum. These results are consistent with, and support, our model for folding of the Ca2(+)-ATPase (Brandl, C. J., Korczak, B., Green, N. M., and MacLennan, D. H. (1986) Cell 44, 597-607) in which residues 657-672 were proposed to form part of the cytoplasmic nucleotide binding domain, while residues 870-890 were proposed to form a luminal loop between proposed transmembrane sequences M7 and M8.
Animals↗
Functional consequences of mutations of conserved amino acids in the beta-strand domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.
The sequences Thr-Gly-Glu-Ser184 and Asp-Gln-Ser178 and individual residues Asp149, Asp157, and Asp162 in the sarcoplasmic reticulum Ca2(+)-ATPase are highly conserved throughout the family of cation-transporting ATPases. Mutant Thr181----Ala, Gly182----Ala, Glu183----Ala, and Glu183----Gln, created by in vitro mutagenesis, were devoid of Ca2+ transport activity. None of these mutations, however, affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+, indicating that the high affinity Ca2(+)-binding sites and the nucleotide-binding sites were intact. In each of these mutants, the ADP-sensitive phosphoenzyme intermediate (E1P) decayed to the ADP-insensitive form (E2P) very slowly relative to the wild-type enzyme, whereas E2P decayed at a rate similar to that of the wild-type enzyme. Thus, the inability of the mutants to transport Ca2+ was accounted for by an apparent block of the transport reaction at the E1P to E2P conformational transition. These results suggest that Thr181, Gly182, and Glu183 play essential roles in the conformational change between E1P and E2P. Mutation of Ser184, Asp157, or Ser178 had little or no effect on either Ca2+ transport activity or expression. Mutations of Asp149, Asp162, and Gln177, however, were poorly expressed. Where expression could be measured, in mutations to Asp162 and Gln177, Ca2+ transport activity was essentially equivalent to that of the wild-type enzyme.
Amino Acid Sequence↗
Functional consequences of alterations to polar amino acids located in the transmembrane domain of the Ca2(+)-ATPase of sarcoplasmic reticulum.
Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 (ligands 1 to 6, respectively) appear to form the high affinity Ca2(+)-binding sites of the Ca2(+)-ATPase. The plasticity of the Ca2(+)-binding sites was tested by separate replacement of each of the ligands with a structurally similar oxygen-containing residue using site-specific mutagenesis. Mutant cDNAs were transfected into COS-1 cells, and ATP-dependent Ca2+ transport or partial reactions were studied in microsomes containing the expressed Ca2(+)-ATPases. In most cases where amino acid substitutions were carried out, the expressed enzymes lacked Ca2+ transport function and Ca2(+)-dependent phosphorylation by ATP. Furthermore, the mutant enzymes were phosphorylated by inorganic phosphate, even in the presence of Ca2+, which inhibits phosphorylation of the wild-type enzyme possessing intact Ca2(+)-binding sites. On mutant, however, containing an isosteric replacement of Glu by Gln at ligand 6, exhibited wild-type levels of Ca2+ transport activity and Ca2+ affinity. Two mutants exhibited properties consistent with a reduction in Ca2+ affinity. In the mutant in which Thr was replaced by Ser at ligand 4, Ca2+ transport activity was 70% of wild-type, while half-maximal activation by Ca2+ occurred at 0.8 microM as compared to 0.3 microM for the wild-type enzyme. In the mutant Glu309----Asp at ligand 1, Ca2+ transport activity was lost, but Ca2(+)-activated phosphorylation by ATP was retained. The concentration of Ca2+ required to activate phosphorylation was increased about 10-fold, however, compared to wild type. These results support our hypothesis that ligands 1 to 6, believed to reside within the transmembrane domain, interact with Ca2+ ions during the transport process. The roles of 12 other oxygen-containing residues and of His278 located in the transmembrane domain were also examined by mutation. Although the oxygen-containing side chains of these residues are potential Ca2+ ligands, their replacement with nonpolar amino acids did not abolish Ca2+ transport function, leading to the conclusion that they are not essential ligands for high affinity Ca2+ binding by the Ca2+ pump.
Amino Acid Sequence↗
Functional consequences of proline mutations in the cytoplasmic and transmembrane sectors of the Ca2(+)-ATPase of sarcoplasmic reticulum.
Site-specific mutagenesis was used to investigate whether Pro160, Pro195, Pro308, Pro312, Pro803, and Pro812 play essential roles in the function of the sarcoplasmic reticulum Ca2(+)-ATPase. All six prolines were substituted with alanine; and in addition, Pro308 was replaced by glycine and Pro312 by glycine as well as by leucine. Mutant cDNAs were expressed in COS-1 cells, and mutant Ca2(+)-ATPases located in the isolated microsomal fraction were examined with respect to Ca2+ uptake activity, Ca2+ dependence of phosphorylation from ATP, and the kinetic properties of the phosphoenzyme intermediates formed from both ATP and Pi. The enzymatic cycle was little affected by substitution of Pro160, Pro195, and Pro812, which are located in the cytoplasmic domain; but replacement of Pro308, Pro312, and Pro803, in the putative transmembrane helices, had a profound impact on the function of the enzyme. All mutations of Pro308 and Pro803 led to ATPases which were characterized by a reduced affinity for Ca2+. These prolines may therefore be involved in the structure of the high affinity Ca2(+)-binding sites in the enzyme. Substitution of Pro312 with alanine or glycine gave rise to mutants unable to transport Ca2+ even though their apparent affinities for Ca2+ in the phosphorylation reaction with ATP were increased. In these enzymes, the ADP-sensitive phosphoenzyme intermediate was stable for at least 5 min at 0 degrees C, whereas the ADP-insensitive phosphoenzyme intermediate decay at a rate similar to that of the wild type. Thus, the inability to transport Ca2+ could be accounted for by a block of ADP-sensitive to ADP-insensitive phosphoenzyme intermediate conformational transition. In contrast, substitution of Pro312 with leucine gave rise to a mutant enzyme that retained about 7% of the normal Ca2+ transport rate. Phosphoenzyme turnover in this mutant also occurred at a low but significant rate, suggesting that the leucine side chain can substitute to some extent for proline.
Animals↗
Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum.
The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.
Amino Acid Sequence↗
Functional consequences of glutamate, aspartate, glutamine, and asparagine mutations in the stalk sector of the Ca2+-ATPase of sarcoplasmic reticulum.
Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+ transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+ transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+ transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+ transport activity to alterations of Asn111 and Asn114. The mutant Asn114 to alanine retained about 50% activity, whereas the change Asn111 to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+ or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+ uptake observed in the Asn111 and Asn114 mutants was not due to a defect in enzyme activation by Ca2+ or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+ following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+ and to channel it to domains that are involved in enzyme activation and cation translocation. Simultaneous mutation of 4 glutamate residues to alanine in the lumenal loop between transmembrane sequences M1 and M2 did not affect Ca2+ transport activity, indicating that acidic residues in this lumenal loop do not play an essential role in Ca2+ transport. Similarly, mutation of Glu192 and Asp196 in the beta-strand domain between stalk helices 2 and 3 did not affect Ca2+ transport activity, although mutation of Asp196 did diminish expression of the protein.
Amino Acid Sequence↗
Location of high affinity Ca2+-binding sites within the predicted transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase.
Cation pumps bind and translocate ions with the intermediate formation of a phosphoenzyme. In spite of extensive knowledge of the primary and even secondary structures of several of these cation transport enzymes, however, no high affinity cation binding sites have yet been determined. Here we report the use of oligonucleotide-directed, site-specific mutagenesis to identify the amino acids involved in Ca2+ binding in one of these transport enzymes, the Ca2+-ATPase of sarcoplasmic reticulum. Alteration of Glu 309, Glu 771, Asn 796, Thr 799, Asp 800 or Glu 908, each of which is predicted to lie near the centre of the transmembrane domain in putative transmembrane sequences M4, M5, M6 and M8 resulted in complete loss of Ca2+ transport function and of Ca2+-dependent phosphorylation of the enzyme by ATP. Phosphorylation of each of the mutant enzymes with inorganic phosphate was observed, however, even in the presence of Ca2+, which inhibits phosphorylation in the wild-type enzyme possessing an intact high affinity Ca2+-binding site. These results suggest that at least six polar, oxygen-containing residues lying near the centre of the transmembrane domain provide ligands for one or both of the two high affinity Ca2+ binding sites in the Ca2+-ATPase.
Animals↗
Molecular cloning and expression of cDNA encoding the 53,000-dalton glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum.
The 53-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum was purified by lentil lectin affinity chromatography and preparative polyacrylamide gel electrophoresis and partially sequenced. Polyclonal and monoclonal antibodies were raised against the 53-kDa glycoprotein and found to cross-react with the 160-kDa glycoprotein. A combination of antibody and synthetic oligonucleotide screening was used to isolate a cDNA encoding the 53-kDa glycoprotein of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The cDNA encodes a protein of 453 amino acids with Mr of 52,421 and a 19-residue amino-terminal signal sequence. The deduced sequence contains two potential glycosylation sites and is largely hydrophilic. The presence of a glycine-rich sequence in the glycoprotein with homology to mononucleotide binding domains supports earlier observations that the glycoprotein binds ATP with high affinity. Although two sequences appear to be hydrophobic on a hydropathy plot, they are not sufficiently long nor sufficiently hydrophobic to qualify unambiguously as transmembrane sequences. The glycoprotein, like calsequestrin, was shown to be inaccessible to trypsin in intact sarcoplasmic reticulum. It can be eluted from the sarcoplasmic reticulum by extraction with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid under hypotonic conditions. Thus, the glycoprotein appears to be localized entirely in the lumen of the sarcoplasmic reticulum and to be associated with the inner membrane surface through Ca2+-dependent mechanisms. Cotransfection of COS-1 cells with cDNAs encoding the glycoprotein and the Ca2+-ATPase led to expression of both proteins with a common localization in the microsomal fraction. The Ca2+ pumping activity of the microsomes isolated from transfected cells was unaltered by the presence of the glycoprotein. Thus the glycoprotein does not appear to modulate Ca2+-ATPase function.
Adenosine Triphosphate↗
Expression and mutation of Ca2+ ATPases of the sarcoplasmic reticulum.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Isolation of a fourth cysteinyl-containing peptide of the alpha-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence.
The rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the alpha-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the alpha-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene.
Amino Acid Sequence↗
Problems with topical steroids.
Explore the source record for details and available documents.
Administration, Topical↗