KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM

SLUSE FE, EVENS A, Dierks T, DUYCKAERTS C, SLUSEGOFFART CM, KRAMER R (1991)
BIOCHIMICA ET BIOPHYSICA ACTA 1058(3): 329-338.

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Zeitschriftenaufsatz | Veröffentlicht | Englisch
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Abstract / Bemerkung
The homologous exchange of external [C-14]aspartate/internal aspartate catalyzed by the aspartate/glutamate carrier of rat heart mitochondria was investigated using aspartate-loaded, glutamate-depleted mitochondria. An inhibitor-stop technique was developed for kinetic studies by applying pyridoxal phosphate. Direct initial rate determinations from the linear phase of [C-14]aspartate uptake were insufficiently accurate at high external and/or low internal substrate concentrations. Therefore, the full time-course of [C-14]aspartate uptake until reaching isotope equilibrium was fitted by a single exponential function and was used to calculate reliable initial steady-state rates. This method was applied in bisubstrate analyses of the antiport reaction for different external and internal aspartate concentrations. The kinetic patterns obtained in double reciprocal plots showed straight lines converging on the abscissa. This result is consistent with a sequential antiport mechanism. It implies the existence of a catalytic ternary complex that is formed by the translocator and substrate molecules bound from both sides of the membrane. The K(m) values for aspartate were clearly different for the external and the internal sides of the membrane, 216 +/- 23-mu-M and 2.4 +/- 0.5 mM, respectively. These values indicated a definite transmembrane asymmetry of the carrier. The same asymmetry became evident when investigating the isolated protein from bovine heart mitochondria after reconstitution into liposomes. In this case the K(m) values for external and internal aspartate were determined to be 123 +/- 11-mu-M and 2.8 +/- 0.6 mM, respectively. This comparison demonstrates a right-side out orientation of the carrier after insertion into liposomal membranes. The sequential transport mechanism of the aspartate/glutamate carrier, elucidated both in proteoliposomes and in mitochondria, also seems to be a common characteristic of other mitochondrial antiport carriers.
Erscheinungsjahr
Zeitschriftentitel
BIOCHIMICA ET BIOPHYSICA ACTA
Band
1058
Zeitschriftennummer
3
Seite
329-338
ISSN
PUB-ID

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SLUSE FE, EVENS A, Dierks T, DUYCKAERTS C, SLUSEGOFFART CM, KRAMER R. KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA. 1991;1058(3):329-338.
SLUSE, F. E., EVENS, A., Dierks, T., DUYCKAERTS, C., SLUSEGOFFART, C. M., & KRAMER, R. (1991). KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA, 1058(3), 329-338. doi:10.1016/S0005-2728(05)80128-8
SLUSE, F. E., EVENS, A., Dierks, T., DUYCKAERTS, C., SLUSEGOFFART, C. M., and KRAMER, R. (1991). KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA 1058, 329-338.
SLUSE, F.E., et al., 1991. KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA, 1058(3), p 329-338.
F.E. SLUSE, et al., “KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM”, BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1058, 1991, pp. 329-338.
SLUSE, F.E., EVENS, A., Dierks, T., DUYCKAERTS, C., SLUSEGOFFART, C.M., KRAMER, R.: KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM. BIOCHIMICA ET BIOPHYSICA ACTA. 1058, 329-338 (1991).
SLUSE, FE, EVENS, A, Dierks, Thomas, DUYCKAERTS, C, SLUSEGOFFART, CM, and KRAMER, R. “KINETIC-STUDY OF THE ASPARTATE GLUTAMATE CARRIER IN INTACT RAT-HEART MITOCHONDRIA AND COMPARISON WITH A RECONSTITUTED SYSTEM”. BIOCHIMICA ET BIOPHYSICA ACTA 1058.3 (1991): 329-338.

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