Activator-substrate-depletion system

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

A commonly used mass transfer reaction model is presented in Figure 8.1a, where the reaction occurs in the bulk aqueous phase [35, 47]. It is assumed that the substrate dissolved in the organic phase diffuses into the aqueous phase, reaching equilibrium. In the absence of reaction, once equilibrium is achieved, apparent mass transfer ceases. Given the presence of active enzyme, depletion of substrate in the aqueous phase occurs, and the system moves into a new equilibrium. Thus, the overall reaction rate depends both on reaction and mass transfer. 

Measurement of adenyl cyclase activity was a laborious and imprecise procedure until the introduction of the method of Krishna et al. This method is based on the production of labeled cyclic AMP from ATP-a- P. Labeled product is separated from substrate by a combination of ion exchange chromatography and adsorption of substrate to nascent BaS04 precipitate. With appropriate equipment, it is possible to perform 300 adenyl cyclase assays in one day. A complication of the method arises from the fact that adenyl cyclase preparations invariably contain sufficient ATPase activity to deplete rapidly the substrate concentration, thereby shortening the period of time over which adenyl cyclase activity can be observed and precluding even the simplest kinetic analysis. This problem has generally been overcome by addition of an ATP-regenerating system to the adenyl cyclase assay medium . In addition, it has recently been shown that 5 -adenylyl-imidodiphosphate (AMP-PNP) is a substrate for adenyl cyclase but not for ATPase ... 

Unfortunately, It Is difficult to make a direct comparison of our data and those of Justin, et al. since there Is little overlap between the molecular species In their system and those we have used so far. It Is possible, however, that the most active substrate DAG s In these membranes had been depleted and were not replenished In the Isolated system. We know that DAG Is not at Its optimal substrate level In castor bean. On the other hand, such differences In chollnephosphotransferase found In studies of the mammalian enzymes have led to the suggestion of tissue differences. The possibility of artifacts also must be considered. 

The reduction of UQ can be measured by the disappearance of an absorption band at 275 nm. Using this technique, it was shown that adding a substrate such as succinate caused a rapid reduction of essentially all the UQ present in the inner membrane the resulting UQH2 could be reoxidized by the cytochrome system in the presence of 02. To determine whether UQ is a necessary participant in electron transport from succinate to 02, the quinone was removed from mitochondria by selective extraction with an organic solvent. The depleted mitochondria were incapable of respiration but recovered this activity when UQ was added back. 

On the basis of depletion/repletion studies, an intake ofO.2 mgper 1,000kcal is required to maintain normal urinary excretion, but an intake of 0.3 mg per 1,000 kcal is required for a normal transketolase activation coefficient. At low levels of energy intake, there will be a requirement for metabolism of endogenous substrates and to maintain nervous system thiamin triphosphate. 

Jurkat cells treated with staurosporine (STS) were used as a model system for the simultaneous quantification of active caspase-7 and active caspase-3. PARP, a natural substrate for caspase-3 (29,30), is cleaved in apoptotic cells to generate a 23 kDa fragment. PARP was cleaved in the first hour with staurosporine (STS) and full-length PARP was nearly depleted by 3 h (Fig. 5A). The results show that Jurkat cells initiate an immediate and rapid apoptosis response to STS. 

Nitrous oxide is produced as a byproduct in multimillion Ib/year quantities in nylon manufacture worldwide. Currently, there is a great interest toward the utilization of NjO due to the environmentally hazardous nature of this gas with respect to the greenhouse effect and ozone layer depletion. In addition to their ability to utilize dioxygen for catalytic hydrocarbon oxidations, ruthenium porphyrins have been shown to activate nitrous oxide which is an extremely inert molecule and a poor ligand. Groves and Roman have found that N O reacted with Ru"(TMP)(THF)2 in toluene to produce Ru (TMP)(0)2 . trans-dioxoRu(VI) complex can in turn epoxidize a suitable substrate such as tra/js-p-methyl styrene. This system was subsequently shown to be catalytic under appropriate conditions . 

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