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Biomedical subjects

S Marzuki

Publications and source records attributed to S Marzuki.

At least 73 records · Page 4Linked to original sources
PubMed1988

Isolation and characterisation of monoclonal antibodies against hydrophobic membrane subunit 9 of the yeast mitochondrial H+-ATPase.

Five stable lines of myeloma-spleen cell hybrids, producing antibodies against the proteolipid subunit 9 of the yeast mitochondrial H+-ATPase F0-sector, have been isolated by immunizing mice with a proteolipid preparation in the presence of sodium dodecyl sulphate. One of these monoclonal antibodies also reacted with subunit 8 of the enzyme complex indicating a shared epitope. The antibodies did not react with the holo-H+-ATPase, suggesting that their epitopes are shielded by other subunits of the enzyme complex.

Animals↗
PubMed1988

Progression from MERRF to MELAS phenotype in a patient with combined respiratory complex I and IV deficiencies.

Identical twins developed myoclonic epilepsy in their teens. One twin remained mildly affected but the other went on to develop sensorineural deafness and ataxia with lactic acidosis and ragged red fibres leading to a diagnosis of mitochondrial encephalopathy. Multiple stroke-like episodes with hemiparesis followed, indicating progression from a MERRF to a MELAS phenotype. Biochemical studies revealed a severe deficiency of mitochondrial NADH-ubiquinone reductase and a moderate deficiency of cytochrome aa3. Western immunoblotting experiments using polyclonal antibodies raised against human placental cytochrome oxidase identified a similar profile of bands to those seen in controls, supporting the view that cytochrome aa3 deficiency in this case may be a secondary consequence of a failure of assembly related to a severe proximal respiratory chain defect.

Acidosis, Lactic↗
PubMed1988

Protein synthesis in mitochondria isolated from human skeletal muscle. Detection of polymorphism in mitochondrial translation products.

The importance of the mitochondrial protein synthesizing system in the development of functional mitochondria, and thus presumably in the pathogenesis of mitochondrial cytopathies has become apparent in recent years. A procedure has been developed to allow the measurement of the protein synthetic activity in mitochondria isolated from human skeletal muscle biopsy materials. The examination of the mitochondrial protein synthesis products revealed two polymorphic variants with the electrophoretic mobilities in SDS-polyacrylamide gel of a 20/22 kDa and a 45/47 kDa protein. Since functional consideration suggests that these variants are most likely to be associated with conservative amino acid substitutions, the observation indicates that it might be possible to electrophoretically detect certain alterations in the mitochondrial translation products in mitochondrial cytopathies due to mutations in the mitochondrial genome.

Humans↗
PubMed1988

Production of subtype-specific antipeptide antibodies to human interferon-alpha 1 and -alpha 4.

Antibodies that are specific to the human interferon (IFN)-alpha 1 and -alpha 4 subtypes have been produced by immunizing rabbits with two short synthetic peptides, corresponding to residues 99-111 of IFN-alpha 1 and residues 37-50 of IFN-alpha 4, respectively. The IFN-alpha 1 peptide has at least three closely clustered residues that are different from those in the other IFN-alpha subtypes, while the IFN-alpha 4 peptide has only two unique amino acid residues, separated by five common residues. The antibodies raised against the IFN-alpha 1 peptide react with recombinant human IFN-alpha 1 but do not cross-react with recombinant human IFN-alpha 4 or IFN-alpha 2. The antibodies raised against the IFN-alpha 4 peptide react with IFN-alpha 4, cross-react with IFN-alpha 1 but not with IFN-alpha 2; the affinity of the antibodies to IFN-alpha 1, however, is at least 10 times lower than their affinity to IFN-alpha 4.

Animals↗
PubMed1987

Assembly of the mitochondrial ribosomes in a temperature-conditional mutant of Saccharomyces cerevisiae defective in the synthesis of the var1 protein.

An investigation of the role of the var1 protein in the assembly of the yeast mitochondrial ribosomes was carried out in a temperature conditional mutant, strain h56, which contains a mutation (tsv1) just upstream of the structural gene for the var1 protein. The mutation results in a marked decrease in the synthesis of the var1 protein at the permissive temperature of 28 degrees C and an apparently complete absence of var1 synthesis at the restrictive temperature of 36 degrees C. Long-term growth of strain h56 at the non-permissive temperature was found to result in the loss of the small (37 S) ribosomal subunit and the appearance of a novel 30 S ribonucleoparticle. Both the small (37 S) and the large (54 S) mitochondrial ribosomal subunits were found to be assembled in strain h56 for at least 3 h after transfer to the non-permissive temperature.

Fungal Proteins↗
PubMed1987

Mitochondrial studies in Kearns-Sayre syndrome: normal respiratory chain function with absence of a mitochondrial translation product.

Intact mitochondria were isolated from skeletal muscle of two patients with Kearns-Sayre syndrome (retinitis pigmentosa, heart block, chronic external ophthalmoplegia), and mitochondrial protein translation was measured. Mitochondrial protein synthesis was up to 10 times greater than in control subjects and SDS-polyacrylamide gel electrophoresis revealed absence of a translation product with the mobility of a 5 KDa protein. State 3 respiration rates were normal with site 1 and site 2 substrates, suggesting that the absent protein was not a functional subunit of a respiratory chain complex.

Adult↗
PubMed1987

Gas phase sequencing of the proteolipid subunit 9 of the human H+-ATPase in the presence of cetyltrimethylammonium bromide.

The compatibility of cetyltrimethylammonium bromide (CTAB), a quaternary ammonium compound with detergent properties, with gas phase protein sequencing has been examined. Two proteins, one hydrophilic (sperm whale apomyoglobin) and one hydrophobic (the proteolipid subunit 9 of the human mitochondrial H+-ATPase), were successfully sequenced in the presence of this detergent. The presence of CTAB did not affect the repetitive yield during sequencing when compared with polybrene although at high detergent concentrations the initial yield was apparently lower. The sequence of the first forty amino acid residues of the human H+-ATPase proteolipid subunit 9 shows complete homology to the bovine sequence.

Animals↗
PubMed1986

An assessment of the ability of yeast cells to incorporate photolabile fatty acids into their membrane phospholipids in vivo.

The photolabile fatty acids 12-azidooleic, 12-(4-azido-2-nitrophenoxy)oleic, 12-azidolauric and 12-(4-azido-2-nitrophenoxy)lauric are readily taken up in vivo by an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. A low level of the two lauric acid derivatives and none of the two oleic acid derivatives are incorporated into membrane phospholipids. Under certain conditions of growth in the presence of 12-(4-azido-2-nitrophenoxy)oleic acid, the nitrophenylazide group is metabolized to a product that lacks the photolabile azido group.

Drug Stability↗
PubMed1986

Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies.

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.

Antibodies, Fungal↗
PubMed1986

Are all mitochondrial translation products synthesized on membrane-bound ribosomes?

The Arrhenius kinetics for the synthesis of var1, which is a hydrophilic protein of the mitochondrial ribosomes, have been compared to that of other mitochondrial translation products, which are hydrophobic subunits of the respiratory enzyme complexes. Our results indicate that, in the yeast Saccharomyces cerevisiae, the hydrophilic var1 protein is synthesized on membrane-associated mitochondrial ribosomes which cannot be distinguished from those responsible for the synthesis of the hydrophobic mitochondrial translation products.

Fungal Proteins↗
PubMed1985

Defective assembly of the mitochondrial ribosomes in yeast cells grown in the presence of mitochondrial protein synthesis inhibitors.

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in the presence of mitochondrial protein synthesis inhibitors erythromycin and chloramphenicol. Yeast cells grown in the presence of erythromycin (2 mg/ml) do not appear to contain any detectable amounts of the mitochondrial small (37 S) ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S was observed; this particle could be shown to be related to the mitochondrial small ribosomal subunit by two-dimensional gel electrophoretic analysis of its protein components. Since the var1 protein is the only mitochondrial translation product known to be associated with the mitochondrial ribosome, our results suggest that this protein is essential for the assembly of the mature small subunit, and that the var1 protein enters the pathway for the assembly of the small subunit at a late step. In at least one strain of yeast the accumulation of the 30-S particle appears to be very sensitive to catabolite repression. When yeast cells are grown in the presence of chloramphenicol instead of erythromycin, assembly of the small subunit appears to be only partially inhibited, and the presence of the 30-S particle could not be clearly demonstrated. This observation is consistent with the fact that in yeast, chloramphenicol inhibits mitochondrial protein synthesis by about 95% only and that the synthesis of the var1 protein appears to be the least sensitive to this inhibition.

Cell Fractionation↗
PubMed1985

Mitochondrial adenosine triphosphatase in mit- mutants of Saccharomyces cerevisiase with defective subunit 6 of the enzyme complex.

mit- Mutants carrying genetically defined mutations in the oli2 region of the mitochondrial DNA were analysed. Most of these mutants demonstrated either the absence of subunit 6 or its replacement by shorter mitochondrial translation products which could be shown to be structurally related to subunit 6 by using a rabbit anti F1F0-antiserum, and by limited proteolytic mapping of the new mitochondrial translation products. Three representative oli2 mit- strains were analysed for the effects of a grossly altered subunit 6 or of a complete absence of this subunit on the activity and assembly of the H+-ATPase. Our results suggest that this subunit is not required for the assembly of the proton channel of the enzyme complex. Thus, in the absence of subunit 6, the mitochondrial respiratory activities in the oli2 mutants were found to be still sensitive to oligomycin, a specific inhibitor of the H+-ATPase proton channel. Immunoprecipitation of the assembled H+-ATPase subunits from these mutant strains using a monoclonal anti-beta-subunit antibody indicates that subunit 6 is also not essential for the assembly of most F1 subunits to components of the F0 sector.

DNA, Fungal↗
PubMed1985

Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens.

Sera that contained autoantibodies to mitochondria (AMA) by immunofluorescence were examined by immunoblotting for reactivity with mitochondrial polypeptides from various mammalian species, yeast, and E. coli. Mitochondrial polypeptides were separated by polyacrylamide gel electrophoresis, were immobilized on nitrocellulose, and were exposed to sera. The sera tested included 18 AMA-positive sera from patients with primary biliary cirrhosis (PBC), two AMA-positive sera from patients without PBC, and 53 AMA-negative sera. All AMA-positive sera reacted with either one or the other, or usually both of two human mitochondrial polypeptides of 70 kilodalton (kD) and 45 kD. The 53 AMA-negative sera were not reactive with the 70 kD polypeptide, but six reacted with the 45 kD polypeptide. The reactivity of the 70 kD and the 45 kD polypeptide was destroyed by brief exposure to trypsin. The counterpart of the 70 kD reactive polypeptide in human mitochondria was a 65 to 70 kD polypeptide in rat and mouse mitochondria, and a 55 kD polypeptide in yeast and in E. coli. The apparent 45 kD polypeptide was similar in all mitochondrial preparations tested, but no counterpart could be identified in E. coli. Beef heart mitochondria were used to show that the reactive polypeptides were present in a semipurified preparation of the F1 portion of mitochondrial H+ ATPase; however, sera did not react with the beta subunit of ATPase, proposed as a candidate mitochondrial autoantigen. The present molecular characterization of two particular antigens should lead to the more precise identification of these antigens, and also to a clearer insight into the pathogenesis of PBC.

Antibody Specificity↗
PubMed1984

Monoclonal antibodies against subunits of yeast mitochondrial H+-ATPase.

Fourteen stable lines of myeloma-spleen cell hybrids producing antibodies against the mitochondrial H+-ATPase have been isolated. One reacted with the alpha-subunit of the enzyme complex (Mr 56000), nine with the beta-subunit (Mr 54000), and four with a 25 kDa subunit which has not been previously characterized. These antibodies are inhibitory or stimulatory or have no effect upon the enzyme activity. Two of the monoclonal anti-beta-subunit antibodies were found to be particularly effective in immunoprecipitating intact H+-ATPase complex.

Animals↗
PubMed1984

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae.

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.

DNA, Mitochondrial↗
PubMed1984

Biogenesis of mitochondria: defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated.

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H+-ATPase can proceed in a mtDNA-less (rho degree) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H+-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized beta-subunit of the H+-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H+-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.

Antibodies, Monoclonal↗