Enzymic activity, effect dialysis

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

MHbR has been purified from erythrocytes of many species including man (H21, K8, Kll, K12, K14) and from brewers yeast (A6). The 1000-fold purified preparation obtained by Kiese (K14) has been shown, by means of chromatographical studies, to contain FAD. Thus the enzyme has been identified as a flavoprotein. However, the same author could not find any further activating effect of FAD or FMN on active preparations, nor a reactivation of the enzyme previously inactivated by dialysis (K14). 

Due to shifts of the pH-activity profiles, pH values often must be adjusted to obtain the optimal activity of the enzyme under investigation (Maurel and Douzou, 1975). When the above requirements are fulfilled, there is always a residual effect of the cosolvent on enzyme activity. In most cases, such an effect is small compared to the effect of lowering temperature. It must be checked that the effect is instantaneous upon addition of the solvent, independent of time, and fully reversible by infinite dilution or dialysis. If these conditions are not met, one should suspect denaturation. 

There are two types of inhibitors. Reversible inhibitors bind to an enzyme in a reversible fashion and can be removed by dialysis (or dilution) to restore full enzyme activity. Irreversible inhibitors cannot be removed by dialysis and, in effect, permanently deactivate or denature the enzyme. 

While most of the problems in the assay of an activity purified by HPLC are expected and typical of chromatographic work with enzymes, the introduction of this technique into the purification scheme may lead to problems if the fractions obtained from the HPLC purification step are to be measured for enzymatic activity. For example, the salt in each fraction may inhibit any enzymatic activities it contains. Moreover, when ion-exchange HPLC is used the salt concentration will vary in the fractions. Thus it is prudent to study the effects of salt, at the concentration used for elution, on enzyme activity before the chromatography. If the salt is found to be detrimental, it will have to be eliminated or at least reduced in concentration before the chromatography. Removing the salt by dialysis may not be the appropriate way to proceed, however, since the inactivation of enzyme activities is not always reversible. 

Extracorporeal dialysis can be implemented for blood decontamination. Enzymes can be immobilized on dialysis cartridges (Klein and Langer, 1986). In that case, kcJKm has to be as high as possible and the flow rate reduced to increase the efficiency of the reactor. Moreover, accessibility of OP molecules to the enzyme active center must not be altered by the immobilization method or by matrix effects. The enzyme concentration per surface unit has to be maximized to reduce diffusion constraints. First order... 

Removal of zinc by dialysis against 1,10-phenanthroline (Table IV) abolishes activity in proportion to the zinc content 18). On readdition of zinc to two gram atoms per subunit, activity is regained completely. Other metals such as Mn Ca Mg, and Cu are ineflFective in this regard. However, Mg and Mn have an activating effect on the zinc-containing enzyme. 

Meyerhoff and Rechnitz (1976) developed a potentiometric creatinine sensor by inclusion of creatinine iminohydrolase between the gas-permeable membrane of an ammonia electrode and a dialysis membrane. Since the specific activity of the enzyme used was very low, 0.1 U/mg, only 43 mU could be entrapped at the electrode. Therefore the sensor was kinetically controlled and reacted to addition of the enzyme activator tripolyphosphate by an increase in sensitivity from 44 mV to 49 mV per concentration decade and a corresponding decrease of the detection limit. These effects agree with theoretical considerations of reaction-transport coupling. The samples were treated with a cation exchanger to remove endogenous serum ammonia. 

The common lipid abnormalities are hypertriglyceridemia in 30-50% of patients, low serum HDL cholesterol in 50-75% (mostly white patients), and hypercholesterolemia in 20% (C5, H3). Hypertriglyceridemia is usually evident when the BUN has exceeded 50 mg% and is caused by decreased lipoprotein lipase activity. Low enzyme activator concentration (low apoCl 1 apoCl 11 ratio), diminished enzyme synthesis due to insulin resistance, a smaller releasable pool of enzyme due to repeated heparinization, and enzyme inhibition by uremic toxins (e.g., spermidine) have all been invoked to explain the decreased lipoprotein lipase activity (C26). Acetate used in the dialysis bath is also claimed to contribute to hypertriglyceridemia through its conversion to acetyl-Co A, but this effect of acetate is probably insignificant (S4). 

Studies in vitro with rat kidney tissue preparations showed that estrogen sulfates inhibit kynurenine transaminase in a reversible fashion that can be relieved by dialysis (MIO, Mil). Competition of sulfate and phosphate esters of estrogens with PLP for the kynurenine transaminase apoenzyme has been demonstrated (S5). An estrogen effect on this enzyme in vivo was suggested by the finding of higher enzyme activity in the kidneys of adult male rats than in either females or estrogen-treated males (M9, Mil). This difference applied to renal kynurenine transaminase, two-thirds of which is in the soluble fraction and the... 

Action of Salts, Antiseptics, Alkaloids and of Other Organic Substances.—In the action of inorganic salts on peptonization, different factors must be considered. Certain salts precipitate albumin in great quantities but even before the quantity used is sufficient to produce predpitation, these already exert an effect on the albuminoid by changing its sensitiveness towards the proteolytic enzyme. The influence of the salts on the digestion is exerted in this case by an indirect method. It is not that the pepsin has been retarded, but rather that the protein substance has become less susceptible. Thus it is that in the presence of certain quantities of NaCl, pepsin with difficulty digests albumin, but a solution of pepsin with addition of sea salt appears very active after dialysis. 

The B2 protein contains 2 moles of non-heme iron per molecule of enzyme. The iron is bound tightly, but can be removed by dialysis against 8-hydroxyquinoline. The iron-depleted enzyme is inactive, but activity may be restored by addition of iron. The B2 protein has a characteristic absorption peak at 410 m/i, which is attributed to its iron content. Hydroxyurea, which is known to inhibit DNA biosynthesis, evidently achieves this effect through interaction with the B2 protein (7). When the B2 protein is treated with hydroxyurea, the characteristic absorption at 410 m/t disappears, and the resulting loss of enzyme activity parallels the decrease in absorbance at that wavelength. Similar effects are obtained with hydroxylamine or hydrazine. 

Irreversibility of inhibition can be established in a number of ways. Basically, excess inhibitor must be removed from the enzyme to isolate the possible reactivation process and enzyme activity monitored with time to test for any reactivation. Methods include exhaustive dialysis of inhibited enzyme with uninhibited enzyme as a control, removing aU excess inhibitor and allowing time for reactivation, followed by assay for activity. An incubation of enzyme and inhibitor followed by dilution into assay solution will measure spontaneous recovery. The stability of the enzyme adduct to exogenous nucleophiles can be determined by diluting the incubation mixture into a solution containing an exogenous nucleophile, such as S-mercaptoethanol or hydroxylamine. Gel filtration or fast filtration columns also effectively remove inhibitor, and activity assays of the protein fraction can monitor any reactivation of the enzyme-inhibitor complex. 

The effect of edta on alkaline phosphatase from E. coli and on the cobalt(ii) and copper(ii) derivatives of the protein has been studied by the measurement of enzyme activity and bye.s.r. spectroscopy. From dialysis experiments on an edta-contaminated apoenzyme, it was found that the edta binds to the metal-free protein. Furthermore, in the complete absence of edta only two Zn + or Co + ions per enzyme molecule were required for full enzyme activity and it is suggested that reports in the literature that more than two metal (ons are necessary (four is the number commonly quoted) may be explained by varying levels of edta contamination in the enzyme and apoenzyme preparations. The ligand edta also affects the e.s.r. spectrum of copper alkaline phosphatase, thus accounting for the two types of signal reported previously and their different behaviour towards phosphate. 

Reversibility. It is known that the effect of eserine on cholinesterase can be completely reversed by prolonged dialysis against water. On the other hand, it proved impossible to obtain any reversal of the poisoning by the phosphorofluoridate esters (see table below). The enzyme solution (5 ml.) was treated with the inhibitor for 15 min. at 38° 1 ml. was used at once for activity estimation, and the remainder dialysed against running water for 24 hr. in the case of the eserine experiment, 36 hr. in the others. It was clear that the combination between the phosphorofluoridate esters and the enzyme is much firmer than that between eserine and the enzyme. 

Standard tests, dialysis, heat inactivation, and the effects of changing the pH on the catalytic activity of the preparations, were all consistent with the idea that enzymes had the properties ascribed to proteins. Between 1920 and 1930 increasing numbers of enzymes were isolated by Willstatter and partially purified. Assays for purification and the extent achieved were not sufficiently rigorous to exclude the possi-... 

Cloetens (98) dialyzed pig kidney phosphatase against 0.01 M KCN for 6 days and found a considerable loss in activity. However, several minutes preincubation with Mg2+ before assay gave up to 40% recovery of activity. Of a series of metal iqns tested, Zn2+ was the most effective giving 70% recovery. Hofstee investigated the effects of glycine, EDTA, and metal ions on calf intestinal phosphatase (99) and concluded that dialysis against EDTA produced an inactive enzyme. Addition of Zn2+... 

Irreversible inhibition, which is much more important toxicologically, can arise from various causes. In most cases the formation of covalent or other stable bonds or the disruption of the enzyme structure is involved. In these cases the effect cannot be readily reversed in vitro by either dialysis or dilution. The formation of stable inhibitory complexes may involve the prior formation of a reactive intermediate that then interacts with the enzyme. An excellent example of this type of inhibition is the effect of the insecticide synergist piperonyl butoxide (Figure 9.6) on hepatic microsomal monooxygenase activity. This methylenedioxyphenyl compound can form a stable inhibitory complex that blocks CO binding to P450 and also prevents substrate oxidation. This complex results from the formation of a reactive intermediate, which is shown by the fact that the type of inhibition changes from competitive to irreversible as metabolism, in the... 

Enzyme inhibition Many types of molecule exist which are capable of interfering with the activity of an individual enzyme. Any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor. Some enzyme inhibitors are normal body metabolites that inhibit a particular enzyme as part of the normal metabolic control of a pathway. Other inhibitors may be foreign substances, such as drugs or toxins, where the effect of enzyme inhibition could be either therapeutic or, at the other extreme, lethal. Enzyme inhibition may be of two main types irreversible or reversible, with reversible inhibition itself being subdivided into competitive and noncompetitive inhibition. Reversible inhibition can be overcome by removing the inhibitor from the enzyme, for example by dialysis (see Topic B6), but this is not possible for irreversible inhibition, by definition. 

Special conditions the above method is valid for a pure enzyme preparation, but cannot give entirely reliable measurements for impure samples. Interfering reactants in the medium may be allowed for by carrying out recovery experiments with a range of amounts of pure SOD added to the test enzyme preparation. Dialysis of the enzyme preparation will eliminate small molecules that may interfere, like ascorbate, reduced glutathione and catecholamines. The addition of 2 //M cyanide may be used to block peroxidases, which has only a minimal effect on the activity of Cu/Zn-SOD. Alternatively 10-5 M azide may be used to block peroxidases without effect on Cu/Zn-SOD. 

There is a great deal of evidence that deficiency of serotonin (5-hydroxytryptamine) is a factor in depressive illness, and many antidepressant drugs act to decrease its catabolism or enhance its interaction with receptors. A key enzyme involved in the synthesis of serotonin (and the catecholamines) is aromatic amino acid decarboxylase, which is pyridoxal phosphate-dependent. Therefore, it has been suggested that vitamin Be deficiency may result in reduced formation of the neurotransmitters and thus be a factor in the etiology of depression. Conversely, it has been suggested that supplements of vitamin Be may increase aromatic amino acid decarboxylase activity, and increase amine synthesis and have a mood-elevating or antidepressant effect. There is little evidence that vitamin Be deficiency affects the activity of aromatic amino acid decarboxylase. In patients with kidney failure, undergoing renal dialysis, the brain concentration of pyridoxal phosphate falls to about 50% of normal, with no effect on serotonin, catecholamines, or their metabolites (Perry etal., 1985). 

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