The process by which free energy from ATP or an ion gradient is coupled to work - either osmotic work, by a membrane pump, or mechanical work, by a molecular motor - as well as the development of force by a molecular motor, can be explained by mechanisms dependent on substrate binding energy. Coupling involves a reaction sequence that combines the driving and driven reactions and that is controlled at switch points where the mobility of the coupling protein and its specificity in binding and catalysis can be abruptly altered; the altered state is an intermediate (or transition state) in the coupled reaction, the unaltered state an intermediate in the uncoupled reaction (slippage). The balance between these states, which is determined by the increase in substrate binding energy in the transformation, decides the relative importance of the coupled and uncoupled paths. On this basis a general expression for switching may be derived: the tightness of coupling is limited by a ratio of substrate dissociation constants before and after a controlling change in state: Ratecoupled/Rateuncoupled [less than or equal to] Kinitial state/Kfinal state.When binding energy is used in this way to distort the conformation of a protein, mechanical work is done; the work is internal but becomes external if the protein is connected to an external load. External work, force F multiplied by distance d, is then limited by a ratio of substrate dissociation constants before and after the conformational change: W = Fd < RT ln(Kinitial state/Kfinal state).The ratios of dissociation constants, estimated from published values of the force exerted by molecular motors, are > 1 × 104 for myosin and > 2 × 104 for kinesin. From the tightness of coupling of the anion exchange carrier of red cells the ratio of constants for coupling is [greater than or equal to] 4 × 104. These increments can be accounted for by conversion of a surface complex to an enclosed complex, as in chelate formation. The work done by an ATP-driven motor is limited by the free energy of ATP hydrolysis, but the force developed is independent of the ATP reaction, and comes from binding energy with either the driving or driven substrate.

The inhibition constant of L-sorbose efflux (Ki(sorbose)) from human erythrocytes for inhibition by D-glucose increases from 5·15 ± 0·89 to 12·24 ± 1·9 mM on cooling from 50¡C to 30¡C; the Ki(sorbose) of D-mannose increases similarly on cooling. The activation energy Ea(sorbose) of net L-sorbose exit from human erythrocytes is 62·9 ± 3·1 kJ mol-1; but in the co-presence of 5 mM D-glucose Ea(sorbose) is reduced to 41·7 ± 1·6 kJ mol-1 (P < 0·005). These data are consistent with the view that when D-glucose binds to the hexose transporter it leads to an activated transporter state which remains transiently activated after glucose dissociates; if L-sorbose binds to this excited state it is more mobile than otherwise and consequently the apparent Ki(sorbose) of D-glucose is raised. Cooling prolongs the decay time of the activated state; hence the Ki(sorbose) of D-glucose rises as temperature is reduced.

The book, which is primarily a teaching aid aimed at a wide target audience, provides a short introductory course to the principles of molecular biology techniques.The drab appearance and cost of this manual belittle the wealth of easily accessed information presented in it. That, which appears to be yet another turgid introduction to the basics of molecular biology is, in fact, a technical handbook covering the whole spectrum of this complex subject in a simple way. The text provides a translation of the intricate subject of DNA technology and its terminology into everyday language. Each procedure is presented as blocks of information generously illustrated with self-explanatory figures which emphasize each point. The format allows incorporation of ideas and methodology into a teaching programme leading the student to understand not only the complexities of molecular biology but also its wider applications, for example its use in plant technology and medicine.

A number of mitochondrial carrier systems function both in homologous and in heterologous exchange mode, which, in the case of the phosphate carrier is a homologous Pi--Pi- and a heterologous Pi--OH- exchange. In addition, we showed that mitochondrial carriers, e.g. the aspartate/glutamate and the phosphate carrier, can undergo a functional shift from coupled antiport to uncoupled uniport after modification of cysteine residues. In this transport mode a mixture of carrier- and channel-type properties is observed. To address this question on the molecular level, the phosphate carrier from yeast (S. cerevisiae) mitochondria was expressed in E. coli, solubilized, purified and functionally reconstituted. From three cysteine residues present in the yeast phosphate carrier at positions 28, 134 and 300, only one single cysteine residue (C28) was found responsible for the functional switch from antiport to uniport. Upon replacement by a serine residue, this interconversion was blocked. After incorporation of the carrier into giant liposomes, electrophysiological methods (patch clamp) were applied. Under these conditions, a fourth transport mode of the phosphate carrier was observed, namely an action as anion-selective channel, which could be reversibly blocked by phosphate.

The cellular uptake of glucose catalysed by the facilitated glucose transporter (GLUT) family is further regulated by metabolites and hormones, most importantly by insulin. All of the six isoforms known in this family possess a large cytoplasmic domain of divergent amino acid sequence. A body of evidence indicates that this domain is important for GLUT regulation. Exactly how this domain participates in the regulation, however, is not known. A likely possibility is that a specific cellular protein interacts with GLUT at this domain, and thus modulates the function. This putative, glucose transporter binding protein (GTBP) may be an enzyme, or a non-enzymic adaptor or docking protein. Indeed, we have identified several cellular proteins that bind to the cytoplasmic domain of GLUT proteins; these include glyceraldehyde-3-phosphate dehydrogenase, glucokinase, GTBP70, GTBP85, GTBP28 and L-3-hydroxyacyl-CoA dehydrogenase. Some of these GLUT-GTPB interactions are functionally coupled. Whether any of these interactions actually participates in the insulin-induced GLUT regulation is yet to be determined.