Complete amino acid sequence of porcine heart citrate synthase

Bloxham, David P.; Parmelee, David C.; Kumar, Santosh; Walsh, Kenneth A.; Titani, Koiti

doi:10.1021/bi00538a009

Cited by 65 publications

(46 citation statements)

References 40 publications

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“…The cDNA contains an unusually long (403 bp) untranslated 3'-region with a polyadenylation consensus sequence AATAAA (Joshi 1987) at position 1832. The 1413-bp-long open reading flame codes for a mitochondrial citrate-synthase protein with a molecular weight of 52.6 kDa (unprocessed form), which is in accordance with the size reported for citrate synthases from A. thaliana (Unger et al 1989), S. cerevisiae (Suissa et al 1984) and pig (Bloxham et al 1982). A comparison of the deduced amino-acid sequence of the potato citrate synthase with citrate synthases from A. thaliana, pig, S. cerevisiae and E. coli is shown in Fig.…”

Section: Resultssupporting

confidence: 84%

“…The entire amino-acid sequence including the N-terminal end has been determined for the mitochondrial citrate synthase from pig (Bloxham et al 1982). By comparison of the protein sequence with the deduced amino-acid sequence of a cloned pig mitochondrial citrate synthase, a 27-amino-acid-long matrix targeting peptide was identified (Evans et al 1988).…”

Section: Resultsmentioning

confidence: 99%

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Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds

Landsch�tze

M�ller-R�ber

Willmitzer

1995

Planta

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A cDNA clone encoding mitochondrial citrate synthase (EC 4.1.3.7), the first enzyme of the tricarboxylic-acid cycle, was isolated from potato (Solanum tuberosum L.) and expression of the enzyme analyzed. The deduced amino-acid sequence of the potato mitochondrial citrate synthase showed high similarity to known citrate synthases from fungi, mammals and Arabidopsis thaliana. The expression pattern of this clone was determined by Northern blot analysis. Expression was detected in all tissues analyzed. The highest level of expression was found in green flower buds. In photosynthetic tissues, stronger mRNA expression was detected in mature than in immature leaves. This rise in expression with leaf age was accompanied by an increase in citrate-synthase activity. Within flowers, expression was severalfold stronger in anthers than in ovaries, indicating a role of mitochondrial citrate synthase during anther or pollen development. A comparatively low level of transcript was detected in underground heterotrophic tissues, such as stolons, tubers and roots. When tubers were stored at low temperature (4 degrees C), mitochondrial citrate-synthase gene expression increased slightly. From the data obtained, we conclude that expression of the mitochondrial citrate-synthase gene is regulated by developmental and environmental factors. The relatively high expression in leaves is in line with the assumption that mitochondria play an important role in photosynthetically active tissues.

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds

Landsch�tze

M�ller-R�ber

Willmitzer

1995

Planta

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“…The amounts analysed ranged over 0.2-1.5 nmol. At that high sensitivity, values for serine, glutamic acid and glycine tended to be high, as also observed by others [2]. Values for contaminants equal to or less than 0.2 mol/mol are omitted from the tables.…”

Section: Analytical Metliod~supporting

confidence: 66%

A Cysteine Cluster Critical for Flavin Binding in Flavocytochrome b2 from Baker's Yeast

Pompon

Lederer

1983

Eur J Biochem

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We have recently described the addition of 2-keto-3-butynoic acid to flavin-free flavocytochrome bz, a reaction which leads to the loss of flavin-binding capacity ('inactivation') [D. Pompon and F. Lederer (1982) Eur. J . Bioclienz. 12Y,1371. For total inactivation, the extrapolated incorporation value was 0.9 mol reagent/mol subunit. In this work we report the results of sequence studies which elucidate the nature of the modification. The modified protein was cleaved with cyanogen bromide and the peptidcs separated on Sephadex G-I 00 and SP-Sephadex C-25. ''C-labeled peptides were digested with trypsin and chymotrypsin and smaller labeled fragments purified by chromatography on Sephadex G-50 and thin-layer fingerprinting.It is shown that three cysteine residues are fractionally labeled with nearly complete mutual exclusion. Furthermore, a fraction of the modified peptides is found under the form of cross-linked fragments, where two cysteines have added to the same ketobutynoate molecule. Only two of the possible cross-links were found. These results show that the three cysteines are close to one another in space in the flavin-free enzyme and hence probably also in the holoenzyme. These results, combined with those obtained in the affinity labeling reaction of holoenzyine with brornopyruvate [Alliel et al. (1982) Eur. J. Biochrm. 122,553 -5581, show that the three residues are located in or close to the active site. Their possible role is discussed.We recently showed that flavin-free flavocytochrome hZ reacts rapidly with 2-keto-3-butynoate to form a covalent adduct which is n o longer active in the presence of added F M N [l]. This loss of activity appears to result essentially from the loss of flavin binding capacity. The cofactor itself affords protection against inactivation and the native enzyme is not affected under the same conditions. The inactivated enzyme was found to have incorporated about one mol reagent/mol subunit, most probably on a cysteine residue.We report in this paper results of peptide isolation experiments. They establish that indeed the modification affects cysteine, but that three different residues in the sequence are. fractionally labeled. Furthermore we provide evidence that these cysteines are close together in space in the flavin-free en.zyme and hence probably also in the native enzyme. MATERIALS A N D METHODS M a trrialsThe preparation of flavocytochrome hZ and its flavin-free derivative were described in our previous paper Analytical Metliod.~Peptide and protein hydrolyses were carried out at 110 "C in 5.7 M HCI, 0.1 phenol, in evacuated sealed tubes. Amino acid compositions were determined with an LKB 4400 amino acid analyser, operated at absorbance = 0.1 for full scale with a normal ninhydrin system. The recorder of the analyser was used to calculate proline, and the 570-nm channel was recorded on a Delsi 510 recorder coupled with a Delsi ICAP 10 integrator. The amounts analysed ranged over 0.2-1.5 nmol. At that high sensitivity, values for serine, glutamic acid and glycine...

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“…C4 (B. sp. C4) (Schendel et a/., 1992) and pig (Bloxham et a/., 1982). The percentages at the end o f the sequences indicate degrees o f identity t o the C. glutamicum citrate synthase.…”

Section: Inactivation Of the Chromosomal Glta Genementioning

confidence: 99%

Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase

et al. 1994

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~ ~~Citrate synthase catalyses the initial reaction of the citric acid cycle and can therefore be considered as the rate-controlling enzyme for the entry of substrates into the cycle. In Corynebacterium glutamicum, the specific activity of citrate synthase was found to be independent of the growth substrate and of the growth phase. The enzyme was not affected by NADH or 2-oxoglutarate and was only weakly inhibited by ATP (apparent Ki = 10 mM). These results suggest that in C. glutamicum neither the formation nor the activity of citrate synthase is subject to significant regulation. The citrate synthase gene, gltA, was isolated, subcloned on plasmid pJCl and introduced into C. glutamicum.Relative to the wild-type the recombinant strains showed six-to eightfold higher specific citrate synthase activity. The nucleotide sequence of a 3007 bp DNA fragment containing the glfA gene and its flanking regions was determined. The predicted gltA gene product consists of 437 amino acids (M, 48936) and shows up to 497% identity with citrate synthase polypeptides from other organisms. Inactivation of the chromosomal gltA gene by genedirected mutagenesis led to absence of detectable citrate synthase activity and to citrate (or glutamate) auxotrophy, indicating that only one citrate synthase is present in C. glutamicum. Transcriptional analysis by Northern (RNA) hybridization and primer extension experiments revealed that the gltA gene is monocistronic (145 kb mRNA) and that its transcription initiates a t two consecutive G residues located 121 and 120 bp upstream of the translational start.

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Complete amino acid sequence of porcine heart citrate synthase

Cited by 65 publications

References 40 publications

Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds

Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds

A Cysteine Cluster Critical for Flavin Binding in Flavocytochrome b2 from Baker's Yeast

Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase

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