PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE

Dierks T, STAPPEN R, SALENTIN A, KRAMER R (1992)
BIOCHIMICA ET BIOPHYSICA ACTA 1103(1): 13-24.

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Zeitschriftenaufsatz | Veröffentlicht | Englisch
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Abstract / Bemerkung
Upon modification of the reconstituted aspartate/glutamate carrier by various amino acid-reactive chemicals a functional lysine residue at the exofacial binding site was identified. The inactivation of transport function by the lysine-specific reagents pyridoxal phosphate (PLP, IC50 400-mu-M) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS, IC50 300-mu-M) could specifically be suppressed by the substrates aspartate and glutamate; a 50% substrate protection was observed at half-saturation of the external binding site. The same held true for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, IC50 500-mu-M) and diethyl pyrocarbonate (DEPC, IC50 20-mu-M), two reagents known to modify carboxylic or histidinyl side-chains, respectively. EDC, however, turned out to catalyze an acylation of the active site lysine by activating carboxyls that had to be present in the incubation medium. This special mechanism, which was proven by protein labelling using EDC/[C-14]succinate, necessitates a lysine side-chain of high reactivity and low pK, since-the modification had to occur at pH less-than-or-equal-to 6.5, i.e. not too far from the pK of the carboxyl to be activated. All reagents applied, additionally including 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, IC50 10-mu-M), were effective at this pH. Competition experiments revealed interaction of EDC, PLP, SITS and probably DIDS at the same active site lysine. For DEPC a lysine modification could not be ruled out. Yet, a model comprising a histidine juxtaposed to the lysine seems to be appropriate.
Erscheinungsjahr
Zeitschriftentitel
BIOCHIMICA ET BIOPHYSICA ACTA
Band
1103
Zeitschriftennummer
1
Seite
13-24
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Dierks T, STAPPEN R, SALENTIN A, KRAMER R. PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE. BIOCHIMICA ET BIOPHYSICA ACTA. 1992;1103(1):13-24.
Dierks, T., STAPPEN, R., SALENTIN, A., & KRAMER, R. (1992). PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE. BIOCHIMICA ET BIOPHYSICA ACTA, 1103(1), 13-24. doi:10.1016/0005-2736(92)90052-N
Dierks, T., STAPPEN, R., SALENTIN, A., and KRAMER, R. (1992). PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE. BIOCHIMICA ET BIOPHYSICA ACTA 1103, 13-24.
Dierks, T., et al., 1992. PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE. BIOCHIMICA ET BIOPHYSICA ACTA, 1103(1), p 13-24.
T. Dierks, et al., “PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE”, BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1103, 1992, pp. 13-24.
Dierks, T., STAPPEN, R., SALENTIN, A., KRAMER, R.: PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE. BIOCHIMICA ET BIOPHYSICA ACTA. 1103, 13-24 (1992).
Dierks, Thomas, STAPPEN, R, SALENTIN, A, and KRAMER, R. “PROBING THE ACTIVE-SITE OF THE RECONSTITUTED ASPARTATE GLUTAMATE CARRIER FROM BOVINE HEART-MITOCHONDRIA - CARBODIIMIDE-CATALYZED ACYLATION OF A FUNCTIONAL LYSINE RESIDUE”. BIOCHIMICA ET BIOPHYSICA ACTA 1103.1 (1992): 13-24.

7 Zitationen in Europe PMC

Daten bereitgestellt von Europe PubMed Central.

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PMID: 19002576
Mitochondrial transporters as novel targets for intracellular calcium signaling.
Satrústegui J, Pardo B, Del Arco A., Physiol Rev 87(1), 2007
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