A lactate-guided conditioning programme to improve endurance performance.

Blood lactate measurements are used widely in horses to assess athletic performance, but there are few published data on using lactate as a guide for endurance improvement. The velocity at which blood lactate concentration ([LA]) of 4 mmol/l is reached (v4) is widely used to determine fitness. In an earlier study of v4, exercise at low intensities for longer duration was more effective at improving endurance than shorter, higher intensities. However, the prescription was unchanged during the study (6 weeks). We hypothesised that, to produce greater improvement in v4 in the same time frame, it is necessary to adjust conditioning regimes more frequently. Six horses underwent an initial treadmill-based standard exercise test (SET). A regression analysis [LA]-speed relationship was used to calculate v2. Horses were then conditioned for 45 min at their calculated v2 3 times/week (6% incline). Every 2 weeks the horses performed an SET to evaluate the v2 conditioning protocol which ended with SET 4. Mean v4 increased 17% from SET 1 to SET 4 (P = 0.003; 5.8 +/- 03 to 6.8 +/- 0.4 m/s). This study demonstrates that a lactate-guided conditioning programme can significantly enhance endurance performance over a 6-week time peroid when the conditioning protocol is adjusted every 2 weeks based on v4 improvement.

Self-catalysed affinity labeling of Q beta replicase.

The spatial neighbourhood of the active center of Q beta replicase can be selectively modified by the method of self-catalysed affinity labeling. In the template-directed, mainly intramolecular enzymatic catalysis, the product [32P]GpG becomes specifically attached to the beta subunit. Using limited digestion of the radioactively labeled polypeptide by cyanogen bromide or N-chlorosuccinimide, we have mapped the attachment site to the region of subunit beta between Trp93 and Met130. Under our reaction conditions, Lys95 is the amino acid most likely to be modified, suggesting that Lys95 lies near the nucleotide binding site in the active center.

Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius.

RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride. The modified protein catalyzes the labeling of its own largest subunit when incubated with [alpha-33P]UTP in the presence of poly[d(A-T)]. On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit. In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerase. This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution.

Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase.

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.