Biomedical subjects
Publications and source records attributed to A R Walmsley.
Myosin from molluscan adductor muscle is regulated directly by Ca2+ binding. In the absence of Ca2+ the ATPase activity is greatly inhibited. We review the application of transient kinetic methods to this system and show how they can be simple to perform and less ambiguous than steady-state methods.
The ATPase activity of scallop (Pecten maximus) striated adductor myosin and heavy meromyosin (HMM) have been investigated as a function of [Ca2+] using formycin triphosphate (FTP) as a fluorescent ATP analogue. The FTPase activity of the regulated fraction of these preparations was activated steeply over the range of 0.1 to 1 microM [Ca2+], implying the existence of a form of cooperativity that is intrinsic to the myosin heads. In addition to the previously characterised heterogeneity with respect to an unregulated fraction, the regulated fraction of HMM was resolved into two populations whose activities showed a slightly different dependency on [Ca2+]. This was revealed unambiguously at intermediate levels of activation where, in some experiments, the product release rate constants differed for the two populations by more than fivefold. At maximum relaxation or maximum activation, these rate constants differed by two- to three-fold and were not clearly resolved by the multiexponential fitting procedure. The populations might arise as a consequence of isoenzymes, modification during preparation or slowly interconverting conformers; Ca2+ binding itself being a rapid equilibrium process in both populations. FTP turnover by myosin could not be analysed in such detail because of the technical problems of measuring the fluorescence of a suspension of filaments, but the rates of the elementary steps appeared similar to those of HMM. The fraction of unregulated molecules in myosin preparations was comparable to that of HMM indicating that if it is a consequence of preparative damage, the modification must occur prior to tryptic digestion.
A method for capturing stopped-flow and other rapid reaction records in logarithmic time, using a microcomputer, is described. Apart from the ability to record processes over several decades in time in a single experiment, the method shows distinct advantages in subsequent nonlinear regression analysis of multiexponential processes. The method is illustrated by a study of the binding of NADPH to dihydrofolate reductase and the reaction between formycin triphosphate and heavy meromyosin.
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Single half-turnovers of the glucose carrier of the human erythrocyte have been measured by recruiting carriers to the outward-facing conformation by (a) pre-exposing cells to extracellular maltose, or (b) pre-warming cells to 38 degrees C, before addition of D-[14C]glucose at 0 degrees C. Based on these experiments estimates of the number of glucose carriers per red cell range from 124,000 to 190,000.
Kinetic data for the transport of glucose and leucine in human red blood cells are fitted to the conventional carrier model and the thermodynamics of the two carrier mechanisms are compared. In the absence of the carried molecule both carriers exist mainly in the inward-facing conformation at low temperatures and the outward-facing conformation at physiological or supra-physiological temperatures, this finding reflecting the strongly endothermic process involved in changing from the inward- to outward-facing forms. Reorientations from inward- to outward-conformations also involve substantial increases in entropy for both carriers. In contrast, substrate binding to the glucose carrier involves little change in enthalpy and an increase in entropy, while leucine binding is strongly exothermic and associated with a decrease in entropy. Application of transition state theory to glucose carrier kinetics reveals that the entropy of formation of the transition state of the carrier is much greater than that for the transition state of the carrier-glucose complex.
A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.
A quenched-flow apparatus is described and applied to measurements of the hydrolysis of 2,4-dinitrophenyl acetate by sodium hydroxide and the entry of D-[U-14C]glucose into human red blood cells at 37 degrees C. Glucose influx into red cells was a saturable process obeying Michaelis-Menten kinetics with a Km for glucose of 6.6 +/- 0.61 mM and a maximum rate for glucose entry under "zero trans" conditions of 20.7 +/- 0.76 mmol (L cell water)-1 s-1. The technique used requires only readily available laboratory equipment and should be easily adaptable to the study of other rapid transport processes.
MULTIFIT, a program in BASIC for implementation on microcomputers, has been developed for non-linear least squares regression fitting of enzyme kinetic, pharmacokinetic and other data to specific models. The program contains a simple procedure for insertion of model equations with up to five parameters (to be fitted) up to 3 independent variables and 1 dependent variable, and can be used to generate a family of programs with pre-set model functions.