Perturbation of yeast 3-phosphoglycerate kinase reaction mixtures with ADP: transient kinetics of formation of ATP from bound 1,3-bisphosphoglycerate.

3-Phosphoglycerate kinase (PGK) is the first ATP-producing enzyme in glycolysis: ADP + 1,3-bisphosphoglycerate (bPG) <--> ATP + 3-phosphoglycerate (PG). Whereas extensive studies have been carried out on its structure, there is less information about its reaction pathway, which is usually studied in the reverse direction because of the instability of bPG. We studied the transients of the PGK reaction by chemical sampling in a rapid quench flow apparatus, using [gamma-(32)P]ATP, in 30% methanol at 4 degrees C to decrease k(cat).

Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M. ADP.Pi states predominate on the cross-bridge cycle.

The ATPases (+/-Ca2+) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different Ea: -Ca2+, 78 and 60 kJ/mol and +Ca2+, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132-3141).

Why choose myofibrils to study muscle myosin ATPase?

Our objective is to propose an overview of the usefulness of skeletal myofibril as an experimental system for studying mechanochemical coupling of skeletal muscles and myosin ATPase activity. The myofibril is a true functional mini-muscle that is able to contract in the presence of ATP. It also contains the machinery necessary for the calcium sensitivity of the contraction.

Evidence that phosphate release is the rate-limiting step on the overall ATPase of psoas myofibrils prevented from shortening by chemical cross-linking.

It has been suggested that the mechanical condition determines the rate-limiting step of the ATPase of the myosin heads in fibers: when fibers are isometrically contracting, the ADP release kinetics are rate-limiting, but as the strain is reduced and the fibers are allowed to shorten, the ADP release kinetics accelerate and P(i) release becomes rate-limiting. We have put this idea to the test with myofibrils as a model because with these both mechanical and chemical kinetic measurements are possible. With relaxed or rapidly shortening myofibrils, P(i) release is rate-limiting and (A)M.

The exo- or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size.

The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed-phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI-TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)(n)C>p, in which C>p indicates a 2',3'-cyclic phosphate.