Mathematical modelling of dynamics and control in metabolic networks. II. Simple dimeric enzymes

The dynamics of enzyme cooperativity are examined by studying a homotropic dimeric enzyme with identical reaction sites, both of which follow irreversible Michaelis-Menten kinetics. The problem is approached via scaling and linearization of the governing mass action kinetic equations. Homotropic interaction between the two sites are found to depend on three dimensionless groups, two for the substrate binding step and one for the chemical transformation. The interaction between the two reaction sites is shown capable of producing dynamic behavior qualitatively different from that of a simple Michaelis-Menten system; when the two sites interact to increase enzymatic activity over that of two independent monomeric enzymes (positive cooperativity) damped oscillatory behavior is possible, and for negative cooperativity in the chemical transformation step a multiplicity of steady states can occur, with one state unstable and leading to runaway behavior. Linear analysis gives significant insight into system dynamics, and their parametric sensitivity, and a way to identify regions of the parameter space where the approximate quasi-stationary and quasi-equilibrium analyses are appropriate.     

A rapid and specific fluorescent activity staining procedure for transamidating enzymes.

A rapid, sensitive, and specific procedure has been developed for detection of transsamidatingenzymes (transglutaminase, e.g., factor XIII) after agarose gel electrophoresis. The technique is based on the transamidase-catalyzed incorporation of the fluorescent monodansylthiacadaverine into casein. The high sensitivity enables detection and characterization of transamidases in blood plasma, platelet, and red blood cell lysate and tissue extracts. The technique can also be combined with crossed immunoelectrophoresis.     

Covalent modification and metabolic control analysis. Modification to the theorems and their application to metabolic systems containing covalently modifiable enzymes

A study of the sensitivity properties of metabolic systems containing covalently modifiable enzymes and cascades has been carried out with the aid of metabolic control analysis. We have considered how the theorems of metabolic control analysis must be modified to take into account covalently modifiable enzymes, and have used these results to investigate the effects of increasing the total amount of modifiable enzyme. The sensitivity of system variables to an effector acting through a covalent-modification cycle has also been investigated.     

A generalization of metabolic control analysis to conditions of no proportionality between activity and concentration of enzymes

The assumption currently considered in the Metabolic Control Theory that velocity of every isolated step is proportional to enzyme concentration, is analysed by considering that in some metabolic systems that condition could not be accomplished. Analysis of the main core of this theory is carried out removing this hypothesis, expressed as "epsilon ei vi is not necessarily equal to one". The results obtained supply a more general formulation of the main theorems of the Control Theory, extending its possible application to more complex metabolic systems.     

Assessment of the genotoxic potential of riboflavin and lumiflavin. A. Effect of metabolic enzymes.

The mutagenic potential of riboflavin and its photodegradation product lumiflavin was evaluated using the umu test, SOS chromotest and Ames Salmonella assay. Both riboflavin and lumiflavin by themselves were found to be non-mutagenic. On treatment with rat liver microsomal enzymes (S9) or caecal cell-free extract (CCE), lumiflavin acquired mutagenicity, while the status of riboflavin remained unaffected. Activation of lumiflavin by metabolic enzymes was found to result in an alteration of its spectral characteristics.     

The metabolic pathway collection from EMP: the enzymes and metabolic pathways database.

The Enzymes and Metabolic Pathways database (EMP) is an encoding of the contents of over 10 000 original publications on the topics of enzymology and metabolism. This large body of information has been transformed into a queryable database. An extraction of over 1800 pictorial representations of metabolic pathways from this collection is freely available on the World Wide Web. We believe that this collection will play an important role in the interpretation of genetic sequence data, as well as offering a meaningful framework for the integration of many other forms of biological data.     

A new method for the rapid identification of genes encoding restriction and modification enzymes.

We have constructed derivatives of Escherichia coli that can be used for the rapid identification of recombinant plasmids encoding DNA restriction enzymes and methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding DNA methyltransferases. The strains of E. coli that we have constructed are temperature-sensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure that is based on the induction at the restrictive temperature of the lacZ gene. Transformants encoding DNA methyltransferase genes are detected on LB agar plates supplemented with X-gal as blue colonies. Using this method, we have cloned a variety of DNA methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema, Pseudomonas, Xanthomonas and Saccharopolyspora.     

Adaptation of red cell enzymes and intermediates in metabolic disorders.

The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.     

Alterations of metabolic genes and metabolites in cancer

Altered metabolic regulation has long been observed in human cancer and broadly used in the clinic for tumor detection. Two recent findings--the direct regulation of metabolic enzymes by frequently mutated cancer genes and frequent mutations of several metabolic enzymes themselves in cancer--have renewed interest in cancer metabolism. Supporting a causative role of altered metabolic enzymes in tumorigenesis, abnormal levels of several metabolites have been found to play a direct role in cancer development. The alteration of metabolic genes and metabolites offer not only new biomarkers for diagnosis and prognosis, but also potential new targets for cancer therapy.     

基于图论的代谢网络中流通代谢物处理新方法

使用图论方法对基因组尺度代谢网络进行途径搜索是目前途径设计中最常用的方法之一.然而,由于流通代谢物(如H+、H2O、CO2和ATP等)的影响,图论搜索得到的途径在生物学上经常是不可行的.虽然前人提出了一些方法对流通代谢物进行处理,但均存在一些问题,目前还没有标准的处理方法.文中基于流通代谢物在反应中作为转移磷酸等功能基...     

A mild procedure for the rapid release of cytoplasmic enzymes from cultured animal cells.

Rapid release of cytosolic enzymes from animal cells in monolayer culture is achieved by treatment of dispersed cells with digitonin. This mild procedure causes efficient cell lysis (>95%) with minimal organelle disruption. A second procedure suitable for isolation of cytosolic enzymes by direct digitonin treatment of cell monolayers has also been developed.