Gad Yagil

Alterations of enzymatic activities during muscle differentiation in vitro

Abstract The activity of creatine phosphokinase, myokinase, and glycogen phosphorylase in muscle cells grown under controlled culture conditions was investigated. Cell fusion and formation of multinucleated muscle fibers were found to be closely associated with a severalfold increase in enzyme activity. The correlation is manifested both in primary cultures and during the differentiation of myogenic cell lines which were maintained for several months under conditions of continuous multiplication. Cytochemical staining for glycogen phosphorylase showed that the increased activity is localized within the multinucleated fibers. Experiments in which the duration of cell fusion was controlled by the concentration of Ca2+ in the nutritional medium suggested that the increase in enzyme activity is largely dependent on the continuation of the fusion process. Application of actinomycin D to cultures during the stage of rapid increase in enzymatic activity did not prevent activity from increasing for several hours, whereas application of inhibitors of protein synthesis did inhibit the increase in activity. In the presence of cyclohexamide, the process of cell fusion was almost totally inhibited while in the presence of actinomycin D fusion continued for several hours. These experiments suggest the possibility that the messenger RNA molecules which specify the synthesis of proteins essential for cell fusion and increased enzymatic activity are formed at a developmental stage preceding cell fusion.

Quantitative aspects of protein induction.

Publisher Summary This chapter discusses the formal aspects of varying two basic parameters of enzyme induction—the parameter of time and the parameter of concentration. The application of the equations obtained was tested on a wide range of systems. The induced synthesis of new proteins is a basic event in many cellular processes, including such fundamental phenomena as cellular differentiation, growth control, metabolic regulation, and cellular secretion. Induced formation of new proteins is currently studied in a great variety of systems including bacterial regulation, viral infection in prokaryotic and eukaryotic cells, enzyme adaptation in cultured cells, hormonal and neural effects in various tissues, and model differentiation systems. The complexity of the biochemical process leading to the formation of a particular protein as a result of arrival of an inducing signal becomes increasingly clear as more evidence accumulates. Most detailed studies of the induction process have been carried out with enzymes, because of the ease with which they are assayed in crude cell preparations.

Are altered glucose-6-phosphate dehydrogenase molecules present in aged liver cells?

Abstract The existence of altered molecules of glucose-6-phosphate dehydrogenase in livers of aged mice was examined by three methods: Electrophoresis on acrylamide gels, inactivation rates at elevated temperatures and single dimensional electroimmunodiffusion. Evidence for the existence of an appreciable part of the enzyme in altered form could not be found.

On the structural complexity of simple biosystems

Quantitative formulations of the concepts of structural complexity, order and regularity in biological systems are proposed. Structural complexity is defined as the minimal number of numerical specifications required to describe an object in physical space. A tentative set of rules to calculate the complexity of point systems (like simple or complex molecules) in real space is formulated. The structural complexity of the following molecules and biostructures is calculated in detail: methane, ethane, adenine, the nucleosome and the Tobacco Mosaic Virus. While the total complexity of the structures increases with the number of atomic or molecular elements, the relative complexity is found to be correlated with the coding requirements for the molecule or the molecular assembly. It is proposed to apply complexity analysis for assessing the coding requirements of biological structures, with the aim of determining whether self assembly processes are sufficient for producing particular structures, or whether specific positional information, provided by the genome of the organism, can be expected to participate in the morphogenetic process. A tentative rule for making that distinction is offered.

The stability of some enzymes in cultured cells.

Abstract The effect of inhibitors of protein synthesis on the activity of 3 enzymes in 2 types of cultured cells—KB cells and rat embryo fibroblasts—was studied. It was found that the activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in rat fibroblasts, as well as of glucose-6-phosphate and malate dehydrogenase in KB cells, stays constant for more than 3 days in the presence of either cycloheximide (5–50 μg/ml) or puromycin (0.1–1.0 μg/ml). This means that the three enzymes are stable entities in their environment in cultured cells. The steady state model often employed to describe enzyme regulation in mammalian tissue can thus not apply to these enzymes in cultured cells, and raises some questions regarding the mechanism of their turnover in organs such as the liver.

DNA unwinding in the CYC1 and DED1 yeast promoters

The capacity of promoter DNA of two yeast genes to be unwound was studied. Both promoters, those of the CYC1 and DED1 genes, contain long oligopurine.oligopyrimidine (R.Y) tracts. The two promoters were cloned into negatively supercoiled plasmids, and their sensitivity to single-strand specific nuclease P1 was examined. Extensive P1 cleavage was located within the R.Y tracts, and cleavage sites were mapped. The extent of cleavage was only slightly dependent on P1 concentration, indicating a slow conversion of an intermediate form of DNA into the P1 reactive state. The cleavage required negative supercoiling and was suppressed by NaCl, MgCl2 and spermine. Two-dimensional topoisomer analysis showed that six superhelical turns were opened in the plasmids examined. The results indicate that at sufficient torsional stress, the R.Y tracts can intermittently undergo a transition into an unwound, ready-to-separate state. The oligopurine.oligopyrimidine tracts may thus serve as DNA unwinding centers in the gene promoters where they reside.

The effect of age on the regulation of glucose-6-phosphate dehydrogenase in mouse liver.

Abstract The activity of glucose-6-phosphate dehydrogenase in mouse liver increases up to 8-fold when young mice are shifted from a high-fat to a fatless diet. Similar changes are exhibited by fatty acid synthetase and 6-phosphogluconate dehydrogenase. The extent of induction decreases appreciably beyond 4 months of age and is only occasionally observed in animals beyond one year of age. In contrast, in animals continuously maintained with induced enzyme levels, repression to basal values takes place at all ages. The problem whether the loss of inducibility is related to body lipid accumulation or is due to a fundamental senescent process is discussed. The changes in activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and fatty acid synthetase from birth through weaning to old age are described.

Isotope exchange in biochemical networks application of an electric circuit technique to a biological reaction system

Abstract Electric circuit techniques are employed to derive the kinetic equations for a class of reactions occurring in complex networks as encountered in cellular systems, namely, exchange at equilibrium. The equations connecting equilibrium velocities with kinetic parameters of individual steps of an enzymatic reaction are derived in three ways : by direct chemical considerations, by showing that a form of Kirchhoffs rules is valid for exchange at equilibrium, and by employing the device known in electricity as the delta-star transformation.

Complexity Analysis of a Self-organizing vs. a Template-Directed System

The structural biocomplexity of two viral structures is evaluated: that of the small RNA tobacco mosaic virus (TMV) and that of the larger dsDNA bacteriophage T4. Tobacco mosaic virus was chosen as a paradigm of a self-organizing biostructure, while the T4 represents biostructures where genome directed instructions are essential for the achievement of the correct virion structure. A large difference in complexity values is found: C = 4 for the TMV virion versus C = 117 for the tail part of the T4 virion. The considerable difference in these values indicates a correlation between the structural biocomplexity as defined and the pattern coding requirements of these organisms. It is proposed to utilize complexity analysis for the evaluation of expected genomic contribution to structural specification: the higher the complexity of a structure, the more genomic directions, of known or unknown nature, are likely to be required. The elements of biological complexity evaluation (Yagil, 1985; 1993b) are briefly summarized. A quantitative measure of order (0.93 for the T4 tail fiber) is an additional outcome of the formalism employed.

The use of anti-immunoglobulin for the immunoprecipitation of labeled proteins from tissue homogenates.

Abstract An improved immunochemical procedure for the quantitative isolation of labeled minor proteins from tissue homogenates is worked out and is applied to the isolation of glucose-6-phosphate dehydrogenase from mouse liver. Goat anti-enzyme serum is used as primary reagent, followed by rabbit anti-goat IgG, and not by carrier enzyme as in currently used methods. The resulting immunoprecipitates are analyzed by acrylamide gel electrophoresis, so that only counts in enzyme bands are registered. An equivalent precipitate formed with serum from nonimmunized goat serves as an efficient control for coprecipitation.