H. Gutfreund

THE APPLICATION OF PRESSURE RELAXATION TO THE STUDY OF THE EQUILIBRIUM BETWEEN METARHODOPSIN I AND II FROM BOVINE RETINAS

1. Introduction In the sequence of reactions from rhodopsin to the end products of photo-bleaching, opsin and all-trans retinal, the interconversion of metarhodopsin I to metarhodopsin II (MR I + MR II) is the first step which is on the right time scale to be directly involved in triggering a physiological response. It is also widely accepted that the very marked temperature and pres- sure dependence of this reaction indicates that it involves considerable structural rearrangement [ 121. In different hypotheses for the transduction of infor- mation from rhodopsin to the plasma membrane of the rod cell, after absorption of a photon, the protein opsin is supposed to be involved either in the opening of an ion channel for release of calcium [3] or in the activation of a chain of enzyme reactions [4]. In either case it is of major interest to characterize the transition to find out whether the equilibration MR I .+ MR II involves a single step and whether the structure of the protein or of the lipid environment is principally involved. Clearly the answers to the above questions will come from a long term investigation of rhodopsin from different sources with a range of techniques. In the investigations reported here we have applied the pressure relaxation technique [S ] to enable us to study the kinetics of the equilibration MR I + MR II and to compare the results obtained with those derived from flash spectroscopy on the same material. The results presented here illustrate a number of points. It is of considerable interest that the pressure relaxation technique can be applied to the study of rate processes in a highly organised system - the rod outer segments (ROS). We have shown that the transfore mation MR I & MR II in ROS can be described by a

The use of pressure perturbations to investigate the interaction of rabbit muscle myosin subfragment 1 with actin in the presence of MgADP

A question of fundamental importance in the molecular interpretation of the mechanism of muscle contraction is the evaluation of the degree of bridge formation and its rate of change, during the different states associated with the hydrolysis of ATP by actomysin. The states of the system have been defined using rapid flow and oxygen exchange methods [ 1,2]. Rapid flow techniques have some theoretical and experimental limitations when viscous and turbid solutions have to be mixed prior to observation. Our use of the pressure relaxation technique, developed [3] to study the rates of equilibration of the attachment of myosin, subfragment 1 to actin, is intended to complement the above investigations. 2.1. Proteins F actin was prepared from an acetone powder as in [7]. Concentrations were determined fromI?

Numerical methods and computing in laboratories: from log tables and slide rules to laptop computers during a lifetime.

o r,rn= 11.08 cm-’ [8]. Myosin subfragment 1 was prepared from rabbit skeletal muscle myosin as in [9]. For most experiments the isoenzymes of Sl were not separated in order to maximise the yield of Sl . Concentrations are quoted on the basis ofil4, = 115 000 and

The putative isomerization of the lactate dehydrogenase--NADH complex.

to nm = 7.9 cm-‘.

Keith Dalziel. 24 August 1921-7 January 1994

1. Introduction In previous experiments we showed that proton liberation during the oxidation of ethanol by horse liver alcohol dehydrogenase (LADH) occurred during formation of the binary and ternary enzyme-substrate complexes rather than during the hydride transfer step [l] Furthermore this ionisation change is in some way linked to quenching of protein fluorescence, suggesting a change in the environment of one of the two tryptophans of the enzyme subunits [2,3]. From these experiments and further studies of the pH dependence of protein fluorescence a model was proposed for the conformational states of this enzyme [3,4]. In order to account for the different pK, values for ADP-ribose equilibrium binding (8.9) and the NAD’ binding rate (9.8) Shore et al. [4] proposed that two conformations of the enzyme exist at neutral pH, EH and gH. The latter form showed quenched protein fluorescence, and was linked to the ionization of an enzyme functional group with a pK, less than 9. The scheme proposed to account for this is similar to our current mechanism. By the use of relaxation techniques in the present study, it has been possible to demonstrate directly the existence of these confor- mational states and the pH linkage. Coenzyme binding markedly slows down the relaxation of the confor- mational equilibria. 2. Experimental LADH was prepared by the method of Theorell et al. [5] and the concentrations in terms of one

The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation.

INTRODUCTION The aim of this essay is to provide a record of the changes in the recording and handling of data in scientific laboratories during the years 1936–2006 covering my experience in four different countries (Austria, England, USA, and Germany) (1, 2). This is essentially a personal story of my experience during a career in which I started as a lab boy doing routine analysis and became the Director of a Research Unit in a University. It is a fact of life that many very important achievements of bygone days turned out to be ephemeral. The invention of the wheel has maintained its general application, and the invention of the shovel has not been made entirely redundant by the development of giant diggers. However, many of the devices or developments that I am concerned with here have disappeared from use, as have recollections of the names of their erstwhile distinguished authors. Just ask any scientist below the age of 40 what he would do with a slide rule that was an essential tool in the laboratory for more than 2 centuries, until 50 years ago. Much of the work now performed with novel methods could have been achieved with the older ones, albeit more slowly and requiring greater effort and experience. Some goals could not have been achieved at all. As much of the numerical work is now actually performed in the measuring instruments, the discussion must cover the design and use of a wide range of laboratory equipment. Modern equipment makes many skills redundant and in consequence they atrophy as leg muscles do with the universal ownership of cars. Skills that have been lost are glass blowing, soldering, the use of a lathe and other tools for the construction of simple mechanical equipment, and the modification of electrical circuits. Most experimentalists would now be hard put to use the type of precision balance in common use before WW2 or to match the impedance between a source and a receptor. Not only practical manual skills were lost with automation. Numerical skills got lost first with hand calculators and then with the supply of cookbook programs for data analysis. The latter is the mental equivalent of the electronic black box of instruments used blindly with instruction books. This results in the inability to adapt to changing needs of new applications. The question one must ask is whether the loss of some skills with the development of modern equipment is detrimental to the originality of research. ‘‘Outsourcing,’’ the victory of Master of Business Administration policy over ‘‘In-House’’ collaboration between workshop and scientists is certainly detrimental to originality. The resulting disappearance of mechanical and electrical workshop facilities for general use, and the consequent lack of experience among research workers is partly responsible for the fact that the frequency of really original developments is not keeping up with the very considerable increase in the number of laboratories. It is rare for a significant new idea to be developed without the need for at least some modification in existing equipment and often some totally new device that must be built in-house. A separate essay could be written to describe the skills that have been lost when instrumentation and specialization replaced many occupations of the individual research worker. The availability of automated devices and black box electronic equipment makes it easier to use what is readily available rather than to design and construct something novel that might be more suitable. The expanding market due to the increase in the number of scientific establishments makes the commercial production of an ever-wider range of laboratory equipment for general use economically viable. Competition between instrument companies does encourage development, but only of equipment with a sizable demand. There were also financial consequences when log tables, slide rules, and old fashioned pipettes that had not changed in fashion for centuries were replaced with ever new models of computers and automated sampling devices. Budgets of laboratories have changed from petty cash to large grant supports. Over the last 50 years, there have been fundamental changes in the funding of research in UK Universities. Until well after WW2 all funds went to and were distributed by the head of the department (The Professor). The financial support was minimal. I remember that in 1944, soon after starting as a research student in Cambridge, I needed a heater to dry membranes for This article is dedicated to John Eccleston who died in 2009 at the age of 66. Address correspondence to: Herbert Gutfreund, Somerset House, Chilton Road, Upton, Oxfordshire OX11 9JL, UK. E-mail: h.gutfreund@bristol.ac.uk Received 14 November 2010; accepted 14 November 2010

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

Transient kinetic studies of the mechanism of lactate dehydrogenase were helped considerably by the variety of optical signals available. This permitted the direct observation of the formation and interconversion of intermediates [l] . The formation of the binary complex, E. NADH, from free enzyme and reduced nucleotide can be observed both through the enhancement of nucleotide fluorescence and the quenching of protein fluorescence. Quantitative analysis of equilibrium [2] and kinetic [3] experiments indicated no distinction between binding of NADH to pig heart lactate dehydrogenase as monitored by the two signals, provided the correction for the nonlinearity of protein fluorescence quenching [2] is applied. Everse, Berger and Kaplan reported [4-61 that the reaction of reduced acetylpyridine adenine nucleotide with chicken-heart lactate dehydrogenase proceeds in two steps distinguishable through a second-order protein fluorescence signal and a first-order nucleotide fluorescence signal. The process monitored by nucleotide fluorescence was reported to have a rate constant of about 95 s-l and this was interpreted in terms of a first-order isomerisation, following a second-order binding process. We therefore re-examined the kinetics of the reaction of the pig heart enzyme with NADH to see whether we could detect a rearrangement with this species.

The kinetics of the reaction of nitrophenyl phosphates with alkaline phosphatase from Escherichia coli

It would be interesting to correlate changes in the educational system and consequent career opportunities with the number of Fellows who came via an unconventional path to achieve academic distinction. The fact that Keith Dalziel obtained his qualifications through part-time study while earning his living, first as a junior laboratory assistant and later as a clinical biochemist, meant that he never had the opportunity to live among a community of students. Probably not until he went to Hugo Theorell’s Institute in Stockholm in 1955 and then to Sheffield, as a Sorby Research Fellow of the Royal Society (1958-63), did he work among a community of equals with similar interests (for instance Vincent Massey, F.R.S., and Gregorio Weber), although an account that he gives of his time in the Nuffield Department of Clinical Biochemistry suggests an earlier involvement in discussions with a group of researchers interested in haemoglobin. This surely must have contributed to his being single-minded and independent. Once one has achieved so much all by oneself it becomes more difficult to accept the importance of sharpening one’s wits during lively arguments.