Acyl group transfer by proteases forming acyl-enzyme intermediate: kinetic model analysis

A kinetic model of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate has been analyzed. The kinetic model takes into account the subsequent enzymatic hydrolysis of synthesized peptide (P), and so the kinetic curve for this compound shows a maximum (denoted as p(max)). Particular stress is placed on analyzing the effects of initial concentrations and of kinetic constants on the value of p(max).The analysis has demonstrated that at a given ratio of initial S and N concentrations, p(max) is affected only by (i) the ratio of the second-order rate constants for enzymatic hydrolysis of S and P(alpha) and (ii) the ratio of rate constants for an attack of the acyl-enzyme intermediate by the nucleophile and water (beta). These conclusions apply regardless of the existence of linear inhibition by the components of the reaction mixture. Thus, the kinetically controlled maximum yield of peptide (p(max)) can be calculated a priori from values of alpha and beta that can be estimated experimentally or from reference data. Simple analytical expressions were obtained, allowing a fairly accurate prediction of p(max) for a broad spectrum of S and N initial concentrations.     

Peripheral growth of axons after reduction of the number of nerve cells in sympathetic ganglia

After chemical desympathization in rats only 30% of cells remain in the stellate ganglia compared with the control. In young rats desympathized at the age of 2 months pressor responses of the arterial pressure to asphyxia and to femoral nerve stimulation disappear. At the age of 4 months these reflexes are restored. Investigation of the catecholamine distribution in the organs (heart) by a fluorescence histochemical method in rats aged 4 months showed that the number of nerve fibers giving a positive reaction for noradrenalin at the periphery is greater than at the age of 2 months. Electron-microscopic investigation of neurons suggests that growth of axons takes place in nerve cells that remain viable.Institute of Child and Adolescent Physiology, Academy of Pedagogic Sciences of the USSR. Second Medical Institute, Moscow. Translated from Neirofiziologiya, Vol. 8, No. 1, pp. 84–90, January–February, 1976.     

Association of muscle fiber composition with health and exercise-related traits in athletes and untrained subjects

Skeletal muscle is a heterogenous and metabolically active tissue, the composition of which is associated with multiple traits. The aim of the study was to determine whether there are additional health and exercise-related traits associated with muscle fiber composition in athletes and non-athletes. This study recruited 164 Russian participants (51 endurance and 48 power athletes; 65 controls). Vastus lateralis muscle fiber composition was assessed by immunohistochemistry. Slow-twitch muscle fiber percentage (STMF%) was significantly greater in endurance than power athletes and non-athletes, and in non-athlete females than males. STMF% was positively associated with athletes’ training frequency, non-athletes’ and endurance athletes’ age, endurance athletes’ competition level and chest depth, and power athletes’ training age. STMF% was negatively associated with diastolic blood pressure in power athletes and with systolic blood pressure and reaction time in non-athletes. In all participants, STMF% was positively associated with age, tolerance to long distance exercise, chest depth and fracture incidence, and negatively with systolic blood pressure and resting heart rate. Age, sex and training frequency explained 10.6% and 13.2% of the variance in STMF% in endurance and power athletes, respectively. This is one of the most comprehensive studies involving athletes and untrained subjects and provides novel information concerning associations of increased STMF percentage with lower resting heart rate, better tolerance to long distances, faster reaction time and larger chest depth. On the other hand, the increased percentage of fast-twitch muscle fibers was associated with rare fracture incidence.     

To the question of Förster theory status and applicability to intermolecular energy migration in photosynthesis

The writing of this paper has been driven by a series of remarks made by reviewers of author’s works in several journals. They asserted that the Förster theory of inductive resonance is inapplicable in cases when electronic excitations are delocalized over several molecules, or plainly dismissed the theory as “out of date”. Since this is doubtlessly a question of general importance, this paper offers a detailed analysis of the types of molecular ensembles and conditions where the use of Förster theory is well founded.

     

Short-lived delayed luminescence of photosynthetic organisms. I. Nanosecond afterglows in purple bacteria at low redox potentials.

A combined study of emissions of purple bacteria Rhodospirillum rubrum, Ectothiorhodospira shaposhnikovii and Thiocapsa roseopersicina was performed under conditions of low potential. It has been shown that a considerable part of the emission represents a delayed luminescence with a lifetime of about 5 ns and an activation energy delta E = 0.05 +/- 0.03 eV. Intensity of this delayed luminescence is approximately equal to that of prompt fluorescence. It diminishes as temperature decreases and also as the intermediate acceptor I becomes reduced after prolonged illumination under low potential conditions. This luminescence represents a radiative decay of the intermediate state, PF, and the luminescence activation energy, delta E, reflects the energy barrier between P*-890 and PF. The value of this barrier determined in the present work is much lower than those obtained previously [3,4,26] for the free-energy release during the primary act of charge separation, basing on redox potential techniques. The reason for this discrepancy is discussed. Delayed luminescence in the picosecond time range is predicted to exist under conditions of active photosynthesis as a result of a small (approx. 0.05 eV) energy barrier between PF and the excited singlet state of reaction center bacteriochlorophyll.     

On-line characterization of monoclonal antibody variants by liquid chromatography-mass spectrometry operating in a two-dimensional format

Recombinant monoclonal antibodies (MAbs) have become one of the most rapidly growing classes of biotherapeutics in the treatment of human disease. MAbs are highly heterogeneous proteins, thereby requiring a battery of analytical technologies for their characterization. However, incompatibility between separation and subsequent detection is often encountered. Here we demonstrate the utility of a generic on-line liquid chromatography–mass spectrometry (LC-MS) method operated in a two-dimensional format toward the rapid characterization of MAb charge and size variants. Using a single chromatographic system capable of running two independent gradients, up to six fractions of interest from an ion exchange (IEC) or size exclusion (SEC) separation can be identified by trapping and desalting the fractions onto a series of reversed phase trap cartridges with subsequent on-line analysis by mass spectrometry. Analysis of poorly resolved and low-level peaks in the IEC or SEC profile was facilitated by preconcentrating fractions on the traps using multiple injections. An on-line disulfide reduction step was successfully incorporated into the workflow, allowing more detailed characterization of modified MAbs by providing chain-specific information. The system is fully automated, thereby enabling high-throughput analysis with minimal sample handling. This technology provides rapid data turnaround time, a much needed feature during product characterization and development of multiple biotherapeutic proteins.