Enzymes in Vitro

It is a simple matter to label a pure enzyme, add it to a homogenate or other tissue preparation, and measure loss of activity and enzyme protein as well as the accumulation of products. Unfortunately, the approach has two major drawbacks (1) it is virtually impossible to establish whether the reaction sequence in vitro bears any resemblance 

Inactivation experiments with pure, labeled phosphoenolpyruvate carboxykinase in vitro suggest the following sequence of events (Ballard et al., 1974 Ballard and Hopgood, 1976 Ballard, 1977). 

Native enzyme is inactivated initially without loss of antibody reactivity. The inactivation reaction is catalyzed by a membrane protein present in all liver membranes but at highest specific activity in plasma membranes and at lowest activity in lysosomal membranes. Inactivation is greatly accelerated in the presence of disulfides such as oxidized glutathione or cystine and retarded by thiols. Disulfides on the membrane protein are implicated because treatment of membranes with dithiothreitol in the presence of iodoacetamide destroys the capacity to inactivate phosphoenolpyruvate carboxykinase. This treatment would reduce and fix protein disulfides. Inactivation requires a membrane protein that shows some tissue specificity, since plasma membranes from reticulocytes or erythrocytes are not active, nor are liposomes prepared from the lipids of liver microsomes. 

Inactivation of enzyme is followed by loss of antibody reactivity. 

Proteolytic cleavage occurs subsequently, with the appearance of lower molecular weight fragments. 

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The 17P-hydroxysteroid oxidoreductase enzymes (HSOR) occur as two distinct isoforms (I and II). They are involved in the conversion of estrone to estradiol. The type I enzyme converts estrone to estradiol (also androstenedione to testosterone) and the type II catalyses the reverse reaction. Phytoestrogens have been shown to inhibit both HSOR enzymes in vitro. 

Inhibition of steroid sulphatase and sulphotransferase Steroid sulphotransferase catalyses the addition of sulphate to steroidal compounds whilst steroid sulphatase catalyses the reverse reaction. In vitro studies have demonstrated that a metabolite of genistein, 4-ethylphenol, can inhibit sulphotransferase (Harris et al, 2000). Sulphoconjugates of daidzein have also been found to potently inhibit these enzymes in vitro (Wong and... 

HARA T, MUKUNOKIY, TSUKAMOTO I, MiYOSHi M, HASEGAWA K (1984) Susceptibility of Kintoki bean lectin to digestive enzymes in vitro and its behavior in the digestive organs of mouse in vivo. J Nutr Sci Vitaminol (Tokyo). 30 381-94. 

Bonk, M. et al., Chloroplast import of four carotenoid biosynthetic enzymes in vitro reveals differential fates prior to membrane binding and oligomeric assembly, Eur. J. Biochem. 247, 942, 1997. 

There are many ligands and group-specific reagents that have been demonstrated to alter the properties of H,K-ATPase, and which are not clinically useful. For example, there is a variety of chemicals that have been used in studies on structure-function relations of H,K-ATPase and that inhibit the enzyme in vitro by modification of its amino [49,67,158], sulfhydryl [95,165,166] or carboxyl groups [140]. 

The most likely explanation for these results is that simple phenolics inhibit a very large array of enzymes in vitro. The most likely explanation for the CNS effects of Uncaria rynchophylla would be that indole alkaloids, such as dihydrocorynanteine or hirsutine (32), interact with the central neurotransmission and possibly the serotonin ergic system. 

In combination of this polymerase with purified propionyl-CoA transferase of Clostridium propionicum, a two-enzyme in vitro PHB biosynthesis system was established which allowed the PHB synthesis from (R)-hydroxybutyric acid as substrate [119]. In this way, the PHB synthesis was independent of the consumption of the expensive CoA, and hence PHA could be readily produced in a semipreparative-scale... 

The activity of Dsz enzymes in vitro was reported to be almost equal for substituted DBTs from CO to C6-DBTs [71]. This implies that the variation in activity found with whole cells is probably due to the different rates of substrate transport into the cells. 

The ability of micro-organisms to produce pectic enzymes in vitro constitutes no proof of their pathogenicity. Some micro-organisms produce pectic enzymes on synthetic-nutrient media, but do not always possess the ability to produce them in vivo. An important role is here played by the susceptibility or resistance of the plant to the effect of the pathogen. Production of D-galacturonanase and pectines-terase by Fusarium oxysporum f. lycopersici was found to be much higher on susceptible than on resistant tomato-stems.287 Likewise,... 

In biological systems, therefore, the behavior of Li+ is predicted to be similar to that of Na+ and K+ in some cases, and to that of Mg2+ and Ca2+ in others [12]. Indeed, research has demonstrated numerous systems in which one or more of these cations is normally intrinsically involved, including ion transport pathways and enzyme activities, in which Li+ has mimicked the actions of these cations, sometimes producing inhibitory or stimulatory effects. For example, Li+ can replace Na+ in the ATP-dependent system which controls the transport of Na+ through the endoplasmic reticulum Li+ inhibits the activity of some Mg2+-dependent enzymes in vitro, such as pyruvate kinase and inositol monophosphate phosphatase Li+ affects the activity of some Ca2+-dependent enzymes— it increases the levels of activated Ca2+-ATPase in human erythrocyte membranes ex vivo and inhibits tryptophan hydroxylase. 

Kim, MJ. et al. 2005. High-throughput screening of inhibitory potential of nine cytochrome P450 enzymes in vitro using liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spec-trom. 19 2651. 

Recently, it was shown that the thromboxane receptor itself is a substrate ofcGMP-PK and cAMP-PK in HEK293 cells, HEL cells, or with purified enzymes in vitro. Phosphorylation of its cytoplasmic carboxyterminal domain prevented the thromboxane receptor from coupling to and activating G-proteins [36, 37]. For intact platelets, TxA2 receptor phosphorylation has not yet been shown, however, it would provide another explanation for the inhibition of PLC activation and subsequent intracellular Ca2+ elevation and granule secretion in response to cyclic nucleotides. 

Kus, B., Caldon, C. E., Andorn-Broza, R., and Edwards, A. M. Functional interactions of 13 yeast SCF complexes with a set of yeast F2 enzymes in vitro. Proteins Structure, Function, and Bioinformatics 2004, 54, 455-67. 

While aspirin is equipotent at inhibiting COX-2 and COX-1 enzymes in vitro and ibuprofen demonstrates a sevenfold greater inhibition of COX-1, other NSAIDs appear to have partial COX-2 specificity, particularly meloxicam. A search for COX-2-speciflc inhibitors resulted in promising candidates such as valdecoxib, celecoxib and rofecoxib. A 30-300 higher potency for inhibiting COX-2, than COX-1, suggested the possibility of relief from pain... 

Giri SN, HolUnger MA (1995) Effect of cadmium on lung lysosomal enzymes in vitro. Arch Toxicol 69(5) 341-345... 

Like most trace elements, nickel can activate various enzymes in vitro, but no enzyme has been shown to require nickel, specifically, to be activated. Howevei, mease has been shown to be a nickel metalloenzyme and has been found to contain 6 to 8 atoms of nickel per mole of enzyme (Fishbein et al.. 1976). RNA (ribonucleic add) preparations from diverse sources consistently contain nickel in concentrations many times higher than those found in native materials from which the RNA ts isolated (Wacker-Vallee, 1959 Sunderman, 1965). Nickel may serve to stabilize the ordered structure of RNA. Nickel may have a role in maintaining ribosomal structure (Tal, 1968, 1969). These studies and other information have led to the suggestion that nickel may play a role in nucleic acid and/or protein metabolism. 

AMP is related to certain mental diseases and may be involved in the action of tranquilizers and antidepressant drugs (60). Whether the ability of diuretic agents such as ethacrynic acid and chlorthalidone to inhibit the enzyme in kidney (68) is related to their diuretic action is also not known. It has been suggested that inhibition of diesterase by diazoxide (59) may explain the hyperglycemic activity of this agent. Several materials are known to activate the enzyme. Imidazole produces strong activation of the enzyme from mammalian tissues (36, 38, 42) but not from E. coli (41). It has been reported (61) that insulin activates the beef heart enzyme in vitro, but it is not known if this has relevance... 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

To better understand the health effects of plant phenolic compounds and to better utilize them, it is necessary to know the molecular mechanisms by which plant phenolic compounds induce cytoprotective enzymes. In vitro studies indicated that plant phenolic compounds such as curcumin often inhibited the enzymatic activities of GST, UGT, SULT as well as cytochrome P450s [Oetari et al., 1996], suggesting that the induction of cytoprotective enzyme activities could not be explained by direct interaction with plant phenolic compounds. On the other hand, much evidence indicates that the increased activity of cytoprotective enzymes are mainly attributable to enhanced transcriptional activation and enzyme synthesis [Holtzclaw et al., 2004]. 

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