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M Saraste

Publications and source records attributed to M Saraste.

At least 145 records · Page 8Linked to original sources
PubMed1985

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria.

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.

Amino Acid Sequence↗
PubMed1984

DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS.

The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS. Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E. coli genome. Sequences that are probably transcriptional promoters for unc and phoS can be detected and the identity of the unc promoter has been confirmed by experiments in vitro with RNA polymerase. Upstream of the promoter sequence is an extensive region that appears to be non-coding. Conserved sequences are found that may serve to concentrate RNA polymerase in the vicinity of the unc promoter. Hairpin loop structures resembling known rho-independent transcription termination signals are evident following the unc operon and glmS. The glmS gene encoding the amidotransferase, glucosamine synthetase, has been identified by homology with glutamine 5-phosphoribosylpyrophosphate amidotransferase.

Amidophosphoribosyltransferase↗
PubMed1984

Location of haem-binding sites in the mitochondrial cytochrome b.

Amino acid sequences of 6 mitochondrial cytochromes b are analysed to locate the binding sites of the two protohaems. Four invariant histidines are found in two protein segments which span the membrane. In each, two histidines are separated by 13 residues. This would place them on the same sides of alpha-helices, and the protohaems could be sandwiched as bis-imidazole complexes between the two transmembrane segments. In this model the haems are located in different halves of the bilayer; the Fe-Fe distance is about 20 A.

Amino Acid Sequence↗
PubMed1984

Calorimetric studies of cytochrome oxidase-phospholipid interactions.

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.

Animals↗
PubMed1984

Clinical findings and return to work after heart valve replacement.

One hundred and thirteen patients operated during the years 1971 to 1976, were re-examined at an average of 26.3 months after heart valve replacement. The functional capacity assessed by the NYHA-classification improved in about 40% of the patients. About 80% considered their symptoms and well-being to have improved after the operation. At the re-examination, heart size was most often enlarged in patients with mitral valve replacement. The average work load measured in bicycle ergometer test was higher in patients with aortic valve replacement compared to those with mitral valve replacement. Patients with aortic valve replacements were working more often (54%) than those with mitral valve replacements (37%). The mean age of patients who were working was significantly lower than in patients who were retired. There was a statistically significant relation between the physical working capacity and the working status. The employability assessed by history and clinical findings corresponded well to the actual work situation in individual patients.

Aortic Valve↗
PubMed1982

E. coli F1-ATPase interacts with a membrane protein component of a proton channel.

The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1. To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding. We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder. We propose that the N-terminal segment lies in the membrane and the rest outside. The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology. These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits. Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation. It is possibly also important in biosynthetic assembly.

ATP Synthetase Complexes↗
PubMed1982

Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels. An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda.

An approach to sequencing proteins by the solid-phase method combined with isolation of proteins and polypeptides by gel electrophoresis is described. Mixtures of proteins or polypeptides resulting from digests are fractionated in the presence of dodecylsulphate in polyacrylamide gels. They are detected with Coomassie blue, eluted, selectively reacted with porous glass derivatives and sequenced in their amino-terminal regions with the aid of a new microsequencer. Alternatively they can be analysed or digested with enzymes and fingerprinted. It is a relatively rapid method of purifying proteins for sequence analysis which we have used to provide partial protein sequence data to complement DNA sequences. Nine genes, four from the unc operon of Escherichia coli encoding the alpha, beta, gamma and epsilon subunits of ATP synthase and five for capsid proteins of bacteriophage lambda, have been identified by this method.

Adenosine Triphosphatases↗
PubMed1982

Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold.

The alpha- and beta-subunits of membrane-bound ATP synthase complex bind ATP and ADP: beta contributes to catalytic sites, and alpha may be involved in regulation of ATP synthase activity. The sequences of beta-subunits are highly conserved in Escherichia coli and bovine mitochondria. Also alpha and beta are weakly homologous to each other throughout most of their amino acid sequences, suggesting that they have common functions in catalysis. Related sequences in both alpha and beta and in other enzymes that bind ATP or ADP in catalysis, notably myosin, phosphofructokinase, and adenylate kinase, help to identify regions contributing to an adenine nucleotide binding fold in both ATP synthase subunits.

ATP Synthetase Complexes↗
PubMed1981

The atp operon: nucleotide sequence of the genes for the gamma, beta, and epsilon subunits of Escherichia coli ATP synthase.

The nucleotide sequence of the promoter distal region of the atp (or unc) operon of Escherichia coli has been determined. It encodes the gamma, beta and epsilon subunits of the ATP-synthase complex and includes a noncoding sequence in which transcription of the operon probably terminates. This work completes the nucleotide sequence of the operon which contains nine genes: eight encode structural proteins of the ATP-synthase complex; a ninth, the first in the operon, may be a pilot for assembly. The genes for the alpha and beta subunits have evolved from a common ancestor.

ATP Synthetase Complexes↗
PubMed1981

Quaternary structure of bovine cytochrome oxidase.

A hydrodynamically homogeneous preparation of bovine mitochondrial cytochrome c oxidase can be obtained by anion-exchange chromatography of alkaline-treated enzyme, followed by a gel permeation chromatography step, which further removes some (aggregated) apoprotein. The molecular weight, Mr, of the monodisperse enzyme in Triton X-100 was found to be 210000. This complex is composed of six different polypeptides, with Mr summing up to about 110000 in toto, in a relative one-to-one stoichiometry. Two sets of these subunits constitute the 210000-Mr enzyme complex. In contrast to our earlier report [Saraste, Penttilä, Coggins, and Wikström, FEBS Lett. 114 (1980) 35-38] the 210000-Mr enzyme contains four (and not two) haems A, and therefore represents the dimer of cytochrome aa3. One of the proposed seven subunits, number III, is lacking in this enzyme preparation.

Animals↗
PubMed1980

Spontaneous and polyamine-induced formation of filamentous polymers from soluble fibronectin.

Fibronectin is a high-molecular-weight glycoprotein present in a soluble form in plasma and in other body fluids and as insoluble protein in connective tissue matrix. This study reports that soluble fibronectin is polymerized into filamentous structures and that polyamines stimulate this process and precipitate fibronectin. Fibronectin purified from human plasma under non-denaturing conditions appeared after negative staining as non-globular extended structures in the electron microscope. During storage of purified fibronectin at +4 degrees C, in particular a low ionic strength, increasing amounts of the protein appeared as protein filaments. These filaments had a diameter of 2--3 nm and a length of up to several micrometers. The filaments also formed bundles of variable thickness, apparently through lateral association. These structures could also be visualized by phase-contrast microscopy. Polyamines, at a concentration of 1--5 mM and at a low ionic strength, induced a rapid, extensive polymerization of fibronectin into filamentous structures. The effect increased in the order putrescine less than spermidine less than spermine. Polyamine-induced precipitation of fibronectin was reversible upon removal of the polyamine. Fibronectin secreted by normal and by malignant cells could be fairly selectively precipitated from the culture medium with polyamines. The observed filamentous polymers of soluble fibronectin resemble the filamentous fibronectin-containing pericellular structures in fibroblast cultures and may provide a model for studies on the deposition of fibronectin in matrix form.

Chromatography, Agarose↗