Relaxation methods and enzymology

A quantitative description of end‐plate currents

“…The quantity x(0) was found previously to be 1-67 + 0-04 (s.E.) msec-' (Magleby & Stevens, 1972), a value quite comparable to those found for enzymes (Hammes, 1968a, b;Chock, 1971; also references cited by Gutfreund, 1971). Therefore, U0 must also have a value typical of energy differences between enzyme conformational states.…”

“…It does seem possible that diffusional and hydrolytic losses from the receptor area could be as rapid as required by this view (see Eccles & Jaeger, 1958), and the rates reported for conformational changes in enzymes could easily be as slow as indicated by the approximately 1 msec-1 decay constants observed for the decaying phases of end-plate currents. The most rapid conformational changes reported for enzymes described by scheme (2) occur at rates of around 10 msec-1 (Erman & Hammes, 1966;Holler, Rupley & Hess, 1969;del Rosaria & Hammes, 1970;Hammes & Simplicio, 1970), and range down to rates of about 0.01 msec-1 (Kirschner, Eigen, Bittman & Voigt, 1966;Halford, Bennett, Trenthan & Gutfreund, 1969;Janin & Iwatsubo, 1969); values close to the 1 msec-1 found for end-plate currents seem most common, however (Hammes, 1968 a, b;Chock, 1971; see also references cited in Gutfreund, 1971). Altogether then, the evidence at hand seems to favour the notion that the decay of end-plate currents is determined by the rate of conformational change, and not by the decline of cleft transmitter concentration.…”

“…As the receptor appears to be a membrane-bound protein with considerable specificity in ligand binding (Changeux, Kasai & Lee, 1970;Changeux, Meunier & Huchet, 1971;Miledi, Molinoff & Potter, 1971), it seems most natural to view the interaction of acetylcholine with its receptor as analogous to an enzymatic process. Following the binding of substrate to enzyme, many enzyme-substrate complexes are thought to undergo a conformational change as the first step in the catalytic sequence (Eigen & Hammes, 1963;Hammes, 1968a, b;Chock, 1971;Gutfreund, 1971); by analogy, acetylcholine would bind to its receptor, and the succeeding conformational change would allow ionic transport through the end-plate channels. Thus, k, fl…”

Voltage clamp analysis of acetylcholine produced end‐plate current fluctuations at frog neuromuscular junction

“…For example, we have assumed that the binding of ACh to the receptor is rapid and voltage independent, whereas the subsequent con-formational change is slower and field-dependent; in terms of this model, the physical significance of the cut-off frequency fc of our conductance fluctuation spectra is intimately related to the dipole moment of the hypothetical gating molecule. Guided by the rapid kinetic studies of enzyme-substrate interactions (Eigen & Hammes, 1963;Hammes, 1968b) (Eigen & Hammes, 1963;Hammes 1968a, b;Chock, 1971;Gutfreund, 1971). This, coupled with the suggestive evidence from artificial membrane work in which the individual channel conductance time courses can be observed and always appear as square pulses, lead us to support the discrete conductance hypothesis (Hladky & Haydon, 1970;Bean, 1972;Ehrenstein et al 1970;Gordon & Haydon, 1972; LaTorre, Ehrenstein & Lecar, 1972 Kasai & Changeux (1971); Katz & Miledi (1972) have already discussed reasons for this rather large discrepancy.…”

“…The fast Fourier transform yields spectral estimates S(f) that are distributed according to x2 distribution with standard deviation a = {VAR(S(f))}1 = S(f)/B.T = 0*2 S(f) (Bendat & Piersol, 1971) Desensitization does not affect the form of S(f) During a constant iontophoretic pulse of ACh the post-synaptic depolarization gradually diminishes (Katz & Thesleff, 1957 Eigen & Hammes, 1963;Hammes, 1968a, b;Gutfreund, 1971; and references cited in Chock, 1971 Our theory may therefore be tested not only by evaluating the accuracy of the equation for e.p.c. spectra (see eqn.…”