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N M Green

Publications and source records attributed to N M Green.

At least 37 records · Page 2Linked to original sources
PubMed1990

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection.

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.

Animals↗
PubMed1990

Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.

We have cloned cDNAs encoding the rabbit and human forms of the Ca2+ release channel of sarcoplasmic reticulum. The human cDNA encodes a protein of 5032 amino acids, with a molecular weight of 563,584, which is made without an NH2-terminal signal sequence. Amino acid substitutions between rabbit and human sequences were noted in 163 positions and deletions or insertions in eight regions accounted for additional sequence differences between the two proteins. Analysis of the sequence indicates that 10 potential transmembrane sequences in the COOH-terminal fifth of the molecule and two additional, potential transmembrane sequences nearer to the center of the molecule could contribute to the formation of the Ca2+ conducting pore. The remainder of the molecule is hydrophilic and presumably constitutes the cytoplasmic domain of the protein. A 114-120 amino acid motif is repeated four times in the protein, in residues 841-954, 955-1068, 2725-2844, and 2845-2958 and a 16 amino acid part of the motif is repeated twice more in residues 1344-1359 and 1371-1386. Although the channel is modulated by Ca2+, ATP, and calmodulin, no clear high affinity Ca2(+)-binding domain of the EF hand type and no clear high affinity ATP-binding domain were detected in the primary sequence. An acidic sequence in residues 1872-1923 contains 79% glutamate or aspartate residues and this sequence is a potential low affinity Ca2(+)-binding site. Several potential calmodulin-binding sites were observed in the sequence, in the region 2800 to 3050.

Amino Acid Sequence↗
PubMed1990

Three-dimensional crystals of CaATPase from sarcoplasmic reticulum. Symmetry and molecular packing.

Structural studies of CaATPase from sarcoplasmic reticulum have so far been restricted to low resolution due to the poor order of two-dimensional crystal forms. However, we report that three-dimensional microcrystals of detergent-solubilized CaATPase diffract to 7.2 A in x-ray powder patterns and may therefore provide an opportunity to study CaATPase structure at higher resolutions. In the present study, we have characterized the symmetry and molecular packing of negatively stained crystals by electron microscopy (em). By altering the detergent-to-lipid ratio, different sized crystals were produced, which adhere to an em grid in different orientations. Thus, we obtained micrographs of three different projections and from these determined unit cell dimensions to be 151 X 51 X 158 A and the three-dimensional space group to be C2 with an angle beta very close to 90 degrees; x-ray powder patterns of hydrated, unstained crystals yielded dimensions of 166 X 58 X 164 A. Micrographs from each of two principal projections were averaged to produce two-dimensional density maps. Based on these maps and on the previously determined low-resolution structure of CaATPase, a packing diagram for these three-dimensional crystals is presented and major intermolecular contacts are proposed.

Animals↗
PubMed1989

Evidence for the cytoplasmic location of the N- and C-terminal segments of sarcoplasmic reticulum (Ca2+-Mg2+)-ATPase.

Antibodies were produced against 5 peptides corresponding to segments of the (Ca2+-Mg2+)-ATPase of fast-twitch rabbit skeletal muscle sarcoplasmic reticulum (SR) including the N- and C-terminal regions. With the exception of antibodies directed against the peptide corresponding to residues 567-582 all antibodies bound strongly to the ATPase in intact SR vesicles, indicating that the epitopes were located on the cytoplasmic face of the SR. When the vesicles were disrupted, by solubilisation in SDS, binding of these antibodies was unchanged, further supporting the idea that these epitopes were located on the cytoplasmic face of SR. This is the first demonstration of the location of the N- and C-terminal regions of SR (Ca2+-Mg2+)-ATPase. These observations are discussed in the light of current structural models of the ATPase.

Animals↗
PubMed1989

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↗
PubMed1989

The predicted secondary structures of the nucleotide-binding sites of six cation-transporting ATPases lead to a probable tertiary fold.

Six cation-dependent transporting ATPases have homologous sequences in the region asigned by chemical labelling to nucleotide binding. Comparison of the most highly conserved segments with other nucleotide-binding domains showed that the sequences were consistent with a mononucleotide-binding fold and enabled a number of likely folding topologies to be limited to two or three alternatives. One of these possible folds was topologically equivalent to adenylate kinase; this was taken as a model in which the significance of conserved amino acids was investigated. In this model conserved amino acids were grouped around a postulated ATP-binding cleft, satisfactorily accounting for their degree of conservation.

Adenosine Triphosphatases↗
PubMed1989

The fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twitch soleus muscle.

The major form of calsequestrin in rabbit slow-twitch soleus muscle is shown to be identical to that isolated and cloned from rabbit fast-twitch muscle on the following bases: identity of cDNAs cloned from mRNAs from the two muscle sources; equivalent hybridization of a fast-twitch calsequestrin cDNA probe to mRNAs isolated from fast-twitch and slow-twitch muscles; identity of the 23 amino-terminal amino acids; strong binding of 45Ca2+ in a gel overlay of slow muscle sarcoplasmic reticulum protein to a band at the level of the fast-twitch calsequestrin isoform and only weak binding at the level of the cardiac isoform. No evidence was obtained for developmentally regulated alternative splicing of the calsequestrin transcript in mature slow or fast-twitch muscle.

Animals↗
PubMed1989

Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum.

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2+. The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."

Amino Acid Sequence↗
PubMed1988

Electron microscopy and structural model of human fibronectin receptor.

Highly-purified human fibronectin receptor (a heterodimer of two distinct subunits, alpha and beta) was studied using electron microscopy and a variety of preparative procedures. It was found that the receptor consists of a globular head approximately 80 by 120 A and two tails about 20 A thick and 180-200 A long. The whole complex is approximately 280 A long. At low concentrations of detergent the receptor forms doublets, triplets or rosettes associated with the tails which possess the transmembrane portion of the molecule. Computer-assisted structure prediction using the published amino acid sequence of both subunits showed differences in the secondary structure of the tails, the alpha-tail being rich in beta-strands, the beta-tail having five cysteine-rich repeats analogous to the EGF-like repeats of laminin. Estimates of the length of the tails from the predicted structure conformed well with the dimensions obtained from electron micrographs.

Amino Acid Sequence↗
PubMed1988

Tryptic cleavage inhibits but does not uncouple Ca2+ATPase of sarcoplasmic reticulum.

Sarcoplasmic reticulum Ca2+ATPase is cleaved by trypsin at two sites, T1 and T2. Cleavage at T1 is complete, whereas only about 50% of the Ca2+ATPase is digested at the T2 site. In the absence of Ca2+ ionophor, Ca2+-ATPase activity of the digested enzyme remains virtually unchanged. In the presence of Ca2+ ionophor, however, the calculated specific activity of the doubly cleaved Ca2+ATPase is decreased by about 40%. The decrease in Ca2+ transport activity is much more rapid than cleavage of the T2 site, and could be correlated with an increased leak of Ca2+ from the digested vesicles. We obtained evidence that this leakiness is independent of the digestion of the Ca2+ATPase itself and is presumably due to the digestion of some other components of the sarcoplasmic reticulum vesicles. Examination of steady-state phosphoenzyme levels resulting from phosphorylation by ATP and Pi, or dephosphorylation by ADP or ADP/EGTA revealed no difference between the digested and the undigested Ca2+ATPase indicating no change in the equilibria caused by the T2 cleavage. Analysis of the substrate concentration dependence of the Ca2+ATPase activity also led to the conclusion that the digestion at T2 reduced the Vmax of ATP hydrolysis but leaves the Km unchanged. The above results are consistent with the model that cleavage at the T2 site reduces the turnover rate of the Ca2+ATPase reaction cycle by about 40% by slowing down or altering the rate-limiting step without affecting the equilibrium constants of the examined steps. We found no evidence of true uncoupling of Ca2+ transport from ATP hydrolysis correlated with cleavage at the T2 site.

Adenosine Triphosphate↗
PubMed1988

Structure of the rabbit fast-twitch skeletal muscle Ca2+-ATPase gene.

We have isolated two genomic clones which together encode the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. One of the two 16.5 kilobase (kb) genomic inserts in the lambda phage vector Charon 4A contains 23 exons extending from the polyadenylation site at the 3' end of the ATPase gene to within 38 nucleotides of the translation initiation codon in the 5' exon. An overlapping genomic insert of 16.5 kb contains the remainder of the 5' exon and a further 8 kb of upstream sequence. S1 nuclease mapping and primer extension analysis of the 5' end of the Ca2+-ATPase mRNA indicate that the transcription initiation site is located 185 base pairs (bp) upstream of the translation initiation codon. A "TATA" box (CA-TAAA) was found at position -30 and the sequence CCAAT was found at position -78 relative to the transcription initiation site. In a previous study (Brandl, C. J., de Leon, S., Martin, D. R., and MacLennan, D. H. (1987) J. Biol. Chem. 262, 3768-3774) cDNAs for neonatal and adult forms of the fast-twitch Ca2+-ATPase were shown to encode different carboxyl-terminal sequences, presumably as a result of alternative splicing. We have now found that these different DNA sequences encoding different carboxyl-terminal sequences are located in different exons. Exon boundaries of the Ca2+-ATPase gene did not correlate well with proposed domain boundaries for the Ca2+-ATPase protein. The locations of exon/intron boundaries were only partially conserved between the Ca2+-ATPase gene and a Na+/K+-ATPase gene (Ovchinnikov, Y. A., Monastyrskaya, G. S., Broude, N. E., Allikmets, R. L., Ushkaryov, Y. A., Melkov, A. M., Smirnov, Y. V., Malyshev, I. V., Dulubova, I. E., Petrukhin, K. E., Gryshin, A. V., Sverdlov, V. E., Kiyatkin, N. I., Kostina, M. B., Modyanov, N. N., and Sverdlov, E. D. (1987) FEBS Lett. 213, 73-80) and they did not follow closely the boundaries of amino acid sequences that are highly conserved among a group of ion transport ATPases.

Amino Acid Sequence↗