TY - JOUR
T1 - 13C NMR measurement of flux through alanine aminotransferase by inversion- and saturation-transfer methods
AU - Malloy, Craig R.
AU - Sherry, A. Dean
AU - Nunnally, Ray L.
N1 - Funding Information:
This study was performed during the tenure of a ClinicianGScientist Award of the American Heart Association to C.R.M. with funds contributed in part by the Texas Affiliate and during the tenure of a National Institute of Health Senior Fellowship to A.D.S. (HM6766). Partial support was also provided by grants from the National Institutes of Health (HL27427), the American Heart Association (81-950), and Mal-linckrodt, Inc. (R.L.N.). The authors thank Michael Graham for bringing to their attention the Box-Muller function and Ref. (13), and George McDonald for modification of the NT 300 spectrometer.
PY - 1985/9
Y1 - 1985/9
N2 - Rate constants for the reaction catalyzed by alanine aminotransferase were measured by 13C saturation transfer and inversion transfer between the enriched methyl groups of pyruvate and alanine. At a high enzyme concentration four techniques provided a reasonable estimate of parameters. (1) saturation transfer, (2) nonlinear least-squares fitting of inversion-transfer data, (3) linear least-squares fitting of the initial slopes of inversion-transfer data, and (4) a combination of initial slope and saturation-transfer data. At a lower concentration of enzyme, the saturation-transfer method provided the most accurate determination of rate constants. An inversion-transfer experiment produces a series of measurements of magnetization at various delays after inversion from which the parameters of interest may be determined by nonlinear least-squares fitting. Error in the determined parameters is influenced by the delays at which data are acquired, the number of data points acquired, and error in the measurement of magnetization. In the presence of significant noise in the data, the complete inversion-transfer experiment (inversion of both sites, sequentially) must be analyzed for an accurate determination of the parameters. If the inversion-transfer experiment is performed, an experimental protocol that minimizes error in the parameters may be designed to efficiently acquire inversion-transfer data if an estimate of the parameters and error in measurement of magnetization is available. The most efficient approach is the combination of initial slope data from the inversion-transfer experiment with measurement of equilibrium magnetization during saturation.
AB - Rate constants for the reaction catalyzed by alanine aminotransferase were measured by 13C saturation transfer and inversion transfer between the enriched methyl groups of pyruvate and alanine. At a high enzyme concentration four techniques provided a reasonable estimate of parameters. (1) saturation transfer, (2) nonlinear least-squares fitting of inversion-transfer data, (3) linear least-squares fitting of the initial slopes of inversion-transfer data, and (4) a combination of initial slope and saturation-transfer data. At a lower concentration of enzyme, the saturation-transfer method provided the most accurate determination of rate constants. An inversion-transfer experiment produces a series of measurements of magnetization at various delays after inversion from which the parameters of interest may be determined by nonlinear least-squares fitting. Error in the determined parameters is influenced by the delays at which data are acquired, the number of data points acquired, and error in the measurement of magnetization. In the presence of significant noise in the data, the complete inversion-transfer experiment (inversion of both sites, sequentially) must be analyzed for an accurate determination of the parameters. If the inversion-transfer experiment is performed, an experimental protocol that minimizes error in the parameters may be designed to efficiently acquire inversion-transfer data if an estimate of the parameters and error in measurement of magnetization is available. The most efficient approach is the combination of initial slope data from the inversion-transfer experiment with measurement of equilibrium magnetization during saturation.
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U2 - 10.1016/0022-2364(85)90348-8
DO - 10.1016/0022-2364(85)90348-8
M3 - Article
AN - SCOPUS:10344221124
SN - 0022-2364
VL - 64
SP - 243
EP - 254
JO - Journal of Magnetic Resonance (1969)
JF - Journal of Magnetic Resonance (1969)
IS - 2
ER -