The in vitro hydrolysis by porcine kidney prolidase of the imidodipeptide L-alanyl-L-proline was monitored by using 1H high-resolution NMR spectroscopy. The dipeptide exists as an equilibrium mixture of isomers with cis or trans conformation about the peptide bond. The 13C and 1H NMR spectra of the dipeptide displayed well-resolved resonances for each isomer. Inversion-transfer NMR spectroscopy, with a recently developed pulse sequence, was used with a range of temperatures to calculate the unitary rate constants for the exchange between isomers. A new analytical procedure was introduced for directly obtaining estimates of the unitary rate constants from inversion-transfer data. Arrhenius analysis yielded an activation energy for the isomerization of 87.0 +/- 4.1 kJ mol-1. 1H NMR time courses of the prolidase-catalyzed hydrolysis of L-alanyl-L-proline showed a faster removal of the trans isomer as the [enzyme]/[substrate] ratio was increased. The transient-kinetic information coupled with the steady-state kinetic parameters of the enzyme was used to develop two possible models of the overall hydrolytic reaction. Numerical integration of the relevant differential equations using the experimentally determined rate constants gave simulated progress curves that enabled selection of one of the proposed schemes as being the most likely; this proposal entailed absolute specificity of prolidase for the trans isomer of L-alanyl-L-proline. Finally, on the basis of the present work, and information from the literature, we have proposed a new model of the active site of the enzyme.
Summary.The mechanism by which choline accumulates in erythrocytes during treatment with lithium salts has been elucidated. A component of the study was a kinetic description of erythrocyte phospholipase-D, which catalyses the release of intracellular choline from phospholipids. Apparent steady-state kinetic parameters for calcium ions were determined: K^ (± SD) = 0-6 ± 0'3 mmol/1 aqueous cell volume and V^,^ (± SD) =12 + 4 iimo\/\ packed red blood cells (RBC) min"'. Competitive inhibition of the phospholipase-D by barium ions was also observed. Other information concerning choline and lithium levels and red cell life-time was obtained from the literature. Details of the kinetics were used to develop a comprehensive dynamic model of choline metabolism by erythrocytes. The scheme is as follows; phosphatidylcholine associated with high density lipoproteins exchanges with the erythrocyte membrane phospholipids, the neutral phospholipids undergo two dimensional translational and rotational motion and also flip between each layer of the bilayer thus becoming exposed to an intracellularly-located phospholipase-D, whereupon the choline is hydrolysed and released into the intracellular milieu. A choline transport protein, which is able to be inhibited by lithium, mediates the influx and efflux of choline. The differential equations that describe reactant flux in this scheme were integrated numerically and the choline accumulation profiles under various conditions of transport and enzyme inhibition are presented. Computer solution of the model, by using as input values plasma lithium levels in the upper limit of the therapeutic range, required that the red cell life-time be reduced in order to explain the previously observed negative association between choline and increasing lithium levels.The results of the computer simulations under varying initial conditions of plasma and erythrocyte lithium and choline concentrations permit, for the first time, a comprehensive description of those factors affecting erythrocyte choline levels.
The first application of inversion-recovery spin-echo proton nuclear magnetic resonance spectroscopy to the monitoring of reactions in rat brain preparations is presented. The initial report of the assignment of proton spin-echo nuclear magnetic resonance spectra from rabbit brain homogenates (C. R. Middlehurst et al., J. Neurochem. 42, 878-879, 1984) was used to assist in the assignment of spectra acquired from rat brain homogenates that were obtained from animals killed by cervical fracture or focussed microwave irradiation. Microwave-irradiated brains were divided into four major anatomical regions. Differences in metabolite levels were detected when spectra from fresh tissue and from various regions were compared. The in situ steady-state kinetics of prolidase in whole brain homogenate was determined. The procedure relies on the spectral differences between enzyme substrates and reaction products. The concentration dependence of the rate of hydrolysis of glycyl-L-proline was discribable by the Michaelis-Menten expression with a Michaelis constant of 1.90 mmol L-1 and a maximal velocity of 9.30 mumol min-1 mg-1 protein. The reactions catalysed by glutaminase and acetylcholinesterase in the brain were also monitored.
Proton nuclear magnetic resonance (1H NMR) spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence was used to obtain spectra from rabbit brain homogenate. The instrumental parameters required for the acquisition of spectra together with the assignment of major peaks are given. The rationale and prospectus for the use of this technique for the study of neurochemistry is outlined.
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