New looks and outlooks on physical enzymology

Eigen, Manfred

doi:10.1017/s0033583500000445

Cited by 159 publications

(100 citation statements)

References 12 publications

(15 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the tumble frequency assay, the rate of generation of attractant is too rapid to be rate-limiting and, unlike in shallow gradient conditions, the level of X is not an integrated result of stimuli in a changing environment. This system yields a response independent of rate of delivery of the stimulus and, therefore, the recovery curves measure the relaxation to normal in much the same way as temperature jump relaxation curves (20). A conclusion from our experiments is that the total number of tumbles suppressed, when measured over a period of time that encompasses all effects of the stimulus, would depend only on the amount of chemoreceptor occupancy change.$ The ability of the assay to achieve this separation of the stimulus-response relationship provides a tool for the analytical evaluation of the tumble regulator role.…”

Section: Discussionmentioning

confidence: 99%

Quantitation of the sensory response in bacterial chemotaxis.

Spudich¹,

Koshland²

1975

Proc. Natl. Acad. Sci. U.S.A.

163

View full text Add to dashboard Cite

A quantitative assay for the stimulusresponse relationship in bacterial chemotaxis has been developed by measurement of tumble frequency. Application of the assay has shown an additive relationship between changes in receptor occupancy and recovery times. Tumble suppression is related to the change in receptor occupancy and not to its rate of change. The results can be explained in terms of varying levels of a tumble regulator.Bacteria have a simple sensory system that allows them to respond to stimuli and sense the direction of a chemical gradient. This system has been shown to consist of discrete chemical receptors (1-3), a transmission mechanism that relays the information from the receptors to the flagella (4,5,22), and a motor response that converts this information into regulation of the frequency of tumbling (6-9). Because of its amenability to chemical and genetic manipulation, the bacterial system may provide a tool for the understanding of other more complex sensory systems.The thorough investigation of a sensory system depends on the ability to measure quantitatively the response to a defined stimulus. Quantitative methods have already been developed for the tracking of individual bacteria (7) and the migration of a colony of bacteria in a defined gradient (10). Semiquantitative tools are available in the capillary assay (11) and the response of bacteria to temporal gradients of attractants and repellents (6,8). However, a method was needed for the precise and quantitative analysis of the immediate receptor-response relationship. The temporal gradient experiments (6) appeared to provide a basis for such a method. These experiments showed that sudden increases in attractant concentration or sudden decreases in repellent concentration led to a suppression of the spontaneous tumbling of bacteria and that this smooth swimming response gradually relaxed to the normal tumbling pattern over a period of time (6,8). Moreover, this recovery could be influenced by conditions that altered the chemotactic response, such as methionine starvation or genetic mutation (12).We have converted the qualitative observations into a quantitative method by determining the number of smooth swimming bacteria as a function of time after a temporal gradient stimulation. The relaxation back to the normal behavioral pattern is a simple exponential decay preceded by a slower, more complex rate of recovery. The pattern of the excitation and recovery can be analyzed and shown to be consistent with a change in the concentration of a tumble regulator in the population of bacteria. The technique thus allows the study of changing levels of the tumble regulator and assists the analysis of conditions that affect this regulator. METHODSBacterial Strains and Growth Conditions. All strains used in this study are derivatives of Salmonella typhimurium LT2 and have been described in detail (12). Cultures were grown at 300 on a New Brunswick gyratory shaker in Vogel-Bonner citrate buffer (13) plus 1% glycerol for serine and aspartate exper...

show abstract

Section: Discussionmentioning

confidence: 99%

Quantitation of the sensory response in bacterial chemotaxis.

Spudich¹,

Koshland²

1975

Proc. Natl. Acad. Sci. U.S.A.

163

View full text Add to dashboard Cite

show abstract

“…The former mechanism defines pre-equilibrium (41) and depicts a macromolecule existing in two possible conformations, from which the ligand selects only one, i.e. Scheme 2.…”

Section: Methodsmentioning

confidence: 99%

Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold

Vogt

Chakraborty

2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:The pre-existing equilibrium between open (E*) and closed (E) conformations is a defining feature of the trypsin fold, but its kinetic signatures remain elusive. Results: Kinetics of the E*-E interconversion are determined for protease and zymogen. Conclusion: Protease and zymogen differ in the relative distribution of E* and E. Significance: The role of the E*-E equilibrium in the trypsin fold is elucidated.

show abstract

“…For scheme IV, Eigen (1968) has shown that the sums and products of the reciprocal relaxation times are given by: 25) and in favorable cases these two combinations can be plotted versus ([P]+[L]) to yield all four rate constants (Eigen, 1968). If the relaxation times are sufficiently well separated, these equations can be uncoupled to relate the individual relaxation times to particular steps.…”

Section: Relaxation Methodsmentioning

confidence: 99%