Enzyme activity in the crowded milieu

The cytosol of a cell is a concentrated milieu of a variety of different molecules, including small molecules (salts and metabolites) and macromolecules such as nucleic acids, polysaccharides, proteins and large macromolecular complexes. Macromolecular crowding in the cytosolic environment is proposed to influence various properties of proteins, including substrate binding affinity and enzymatic activity. Here we chose to use the synthetic crowding agent Ficoll, which is commonly used to mimic cytosolic crowding conditions to study the crowding effect on the catalytic properties of glycolytic enzymes, namely phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, and acylphosphatase. We determined the kinetic parameters of these enzymes in the absence and in the presence of the crowding agent. We found that the Michaelis constant, K(m), and the catalytic turnover number, k(cat), of these enzymes are not perturbed by the presence of the crowding agent Ficoll. Our results support earlier findings which suggested that the Michaelis constant of certain enzymes evolved in consonance with the substrate concentration in the cell to allow effective enzyme function in bidirectional pathways. This conclusion is further supported by the analysis of nine other enzymes for which the K(m) values in the presence and absence of crowding agents have been measured.     

Enzyme activity during the metabolism of glycogen

Summary Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii.After incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO₄ (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO₄, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.     

Immunomodulating activity of meningococcal antigens

A preparation of meningococcal antigens (MA) extracted in CaCl2, and containing mostly outer membrane proteins, was strongly mitogenic for murine B lymphocytes. Given to mice in vitro, MA markedly impaired subsequent in vivo T-cell responses of splenocytes. Suppression of normal T splenocytes in vitro occurred with both adherent (Ad) and nonadherent (NA) splenocytes from MA-sensitized mice. B cells were much less affected by the suppression induced by MA, and only Ad cells could convey in vitro the low level impairment of B-cell proliferation. Strong T-cell suppression associated with a B-cell mitogen is also produced by bacillus Calmette-Guérin (BCG) and Corynebacterium parvum. The possible role of these phenomena in meningococcal disease is discussed.     

Chromatographic analysis and immunobiologic properties of tuberculins]

Immunobiologic activities of tuberculin preparations and their components have been comparatively studied using gel filtration and high-performance liquid chromatography (HPLC) techniques. It is shown that high-molecular weight fraction of protein-purified derivate tuberculin (PPD) had higher activity as compared to nonfractionated preparation in skin tests on guinea pigs sensitized with Mycobacterium bovis BCG as well as in enzyme-linked immunosorbent assay with affinity purified rabbit antibodies against PPD. Using the preparative HPLC-technique we failed to isolate a component of PPD having greater tuberculin test potency than nonfractionated preparation.     

Enzyme immunoassays for the detection of microbial antigens and prospects for improved assays

The rapid diagnosis of viral infections is an important tool in the management of patients with infectious diseases. Solid-phase enzyme immunoassays have proved to be useful tools for the direct detection of the antigens of some viruses directly in clinical specimens. Such assays have been particularly useful in the diagnosis of viral infections in the gastrointestinal and respiratory tracts. However, standard solid-phase enzyme immunoassays often do not display sufficient sensitivity for the diagnosis of all cases of viral infections. Techniques which might be utilized to increase the sensitivity of solid-phase immunoassays include the use of monoclonal antibodies to maximize the efficiency of the antigen-antibody interactions and the use of high-turnover enzymes to increase the amount of signal generated by the ensuing enzyme-substrate reactions. In addition, techniques making use of nucleic acid hybridization have a great deal of potential for the accurate detection of viral nucleic acids in human body fluids. The successful application of these techniques to the diagnosis of viral infections could lead to a marked improvement in the care of patients with suspected infectious diseases as well as to a decrease in the transmission of viral infections to high-risk individuals.     

Enzyme activity of cryoprotected myocardium.

The use of in vivo intravenously infused DMSO/glycerol in Krebs-Henseleit buffer solution as a cryoprotectant in rat left ventricles was investigated. Alkaline phosphatase and creatine phosphokinase activities were monitored. Data show inhibition of alkaline phosphatase by the cryoprotectant solution. No significant differences were noted for the CPK activity indicating that it may not be affected by either freezing or the cryoprotectant or both in combination.     

Immunogenic activity of synthetic brucellosis antigens

Experimental data obtained in this investigation indicate that the conjugation of Brucella protective antigen with a polymer carrier essentially increase the immunogenic properties of the antigen. Synthesized vaccinal preparations can be of interest for practical use, as these preparations, while inducing the development of intense immunity, do not impede the diagnosis of brucellosis by serological reactions. The comparative study of the conjugates of Brucella antigen with different carriers shows that the conjugated preparation obtained on the basis of modified dextran possesses high protective potency, which makes it possible to regard this carrier as very promising for further use.     

Is frontalis activity a reliable indicator of the activity in other skeletal muscles?

Multichannel integrated action-potential recordings (called electromyometrograms or EMMGs) from 10 individuals with various functional symptoms and disorders were selected for statistical examination to obtain evidence bearing on the topic in question. Each EMMG recorded the action-potential levels from four regions simultaneously via four channels of integrating differential amplifiers. The subjects sat quietly, awake and alert, with eyes open and head erect. Electrodes were placed over antagonistic muscle groups in the following regions: (1) forehead, (2) jaw-throat, (3) right forearm, (4) left leg. Successive 1-minute action-potential levels from each of these regions were used to calculate within-subject correlations (r) and predictabilities (r ² andR ²) among the different regions. Mean and median correlations (r) and predictabilities (r ² andR ²) for each of the following pairs of regions were found to be weak: (1) forehead and jaw-throat, (2) forehead and forearm, (3) forehead and leg, (4) jaw-throat and forearm, (5) jaw-throat and leg, (6) forearm and leg. The findings support the statement that the frontalis or any of the other regions measured are poor indicators of the amount of activity elsewhere in the skeletal musculature of awake and alert individuals. When combined with basic principles of neuroanatomy and neurophysiology, the findings also support the statement that there is probably no portion of the skeletal musculature that can be used to quantify the activity in other portions.     

Enzyme activity and dynamics of Drosophila development

The length of preadult development is negatively correlated with the activity of a majority of studied enzymes, in adult D. melanogaster and D. subobscura flies. This has been shown when activities of seven enzymes (G6PD, 6PGD, GPD, ADH, HK, ME & IDH) were estimated per mg of protein, or of body mass, in four groups of 6-days old males (50 individuals each, with 3 replications), that had an extremely different preadult development rate. The average activity of studied enzymes is for c. 25% decreased in synchronously grown flies with the longest egg-to-adult development at 21°C and optimal laboratory conditions, compared with those of the same species with the fastest growth. When the group of slowest growing D. subobscura males (20.4±0.1 days) is compared with the fastest D. melanogaster flies (10.6±0.03 days), a decrease of 47% in enzyme activity was observed. Among studied gene-enzyme loci, four in D. subobscura (Gpd, Adh, Me & idh) and one in D. melanogaster (Idh) are monomorphic, which implies an involvement of regulatory genes. Among those of D. melanogaster which are polymorphic, specific combinations of alleles have been determined in fast and slow developed flies, suggesting that interactions of structural genes are also of great importance in the control of two studied fitness characteristics.