Membrane-bound enzymes, inhibition

Plasma levels of 3—5 p.g/mL are obtained two hours after adraiinistration of 200 mg ketoconazole. No accumulation in the bloodstream was noted after a 30-wk treatment with this dose. The half-life is approximately eight hours. When ketoconazole is taken with meals, higher plasma levels are obtained. Distribution studies using radioactive ketoconazole in rats show radioactivity mainly in the Hver and the connective tissue. Radioactivity is also present in the subcutaneous tissue and the sebaceous glands. After one dose of 200 mg in humans, ketoconazole is found in urine, saUva, sebum, and cenimen. Like miconazole, the mode of action is based on inhibition of the cytochrome P-450 dependent biosynthesis of ergosterol. This results in disturbed membrane permeabiUty and membrane-bound enzymes (8,10,23,25). 

There has been considerable discussion regarding the mode of action of the sea cucumber and starfish saponins. Both the triterpene and steroidal glycosides inhibit both Na/K ATPase and Ca/Mg ATPase 06) possibly as a result of their aglycone structures. However, their detergent properties cause membrane disruption which will influence the activity of membrane-bound enzymes such as the ATPases. In investigating the actions of saponins on multilamellar liposomes, it was found that cholesterol serves as the binding site for such saponins and that cholesterol-free lip-somes are not lysed by saponins 107). 

Inside the cytoplasm of the presynaptic neuron the monoamines are exposed to the mitochondrial outer membrane-bound enzyme monoamine oxidase (MAO). MAO breaks the monoamines down into inactive metabolites before they are taken up into the vesicles. However, if MAO is inhibited, then the monoamines enter the vesicles and are available for release. MAO inhibitors, such as moclobemide, have been used in the treatment of depression, since they increase the availability of noradrenaline and serotonin. Selegiline is used for Parkinson s disease, since it raises dopamine levels. 

These zinc-dependent endopeptidases (meprin A [EC 3.4.24.18] and meprin B [EC 3.4.24.63] ) are members of the peptidase family M12A. They catalyze the hydrolysis of peptide bonds in proteins and peptide substrates. Meprin A, a membrane-bound enzyme that has been isolated from mouse and rat kidney and intestinal brush borders as well as salivary ducts, acts preferentially on carboxyl side of hydrophobic amino acyl residues. Meprin A and B are insensitive to inhibition by phosphora-midon and thiorphan. 

Subcellular membrane-bound enzymes activity. Oil, administered to male CFY weanling rats at a dose of 20% for 16 weeks, produced an increase of synaptosomal acetylcholinesterase activity in the coconut oil-fed group. The Mg -adenosine triphosphate (ATPase) activity was similar among all groups in all the brain regions Superoxide production inhibition. Seed oil, administered to rats at a concentration of 8% of diet, produced equivocal effect on macrophages. Capsaicin or curcumin enhanced effect . ... 

Ergosterol, the predominant sterol in yeast cells, plays an important role in membrane fluidity, permeability and the activity of many membrane-bound enzymes. In terpene-treated cells, ergosterol synthesis was strongly inhibited, and a global upregulation of genes associated with the ergosterol biosynthesis pathway was described in response to terpene toxicity [80, 121]. 

Keller and coworkers341 proposed that tunicamyein is a reversible, tight-binding, and, therefore, competitive inhibitor of the GlcNAc 1-P transferase. The association rate-constant was 7 x 104 M s 1 (at 23°). Inhibition can be overcome by increasing the proportion of enzyme, and, because preincubation of the enzyme with UDP-GlcNAc prevented inhibition by tunicamyein,341 some experimental support for competitive inhibition was obtained. The known affinity of the antibiotic for phosphonolipids323 may facilitate its access to the membrane-bound enzyme, but the lipids do not prevent inhibition of the enzyme by tunicamyein.340... 

In general, the strength of substrate binding to membrane-bound enzymes spans a range similar to that encountered with soluble enzymes. The presence of a membrane can either enhance or inhibit substrate binding. The solubilized ATPase and the reconstituted enzyme have only slightly different Michaelis constants. 

Skou had established previously that such anesthetics inhibited certain membrane-bound enzymes found in the membrane fraction isolated from nerve homogenates. He decided to study the endogenous ATPase activity as a possible target for future work, initially thinking that it might be the sodium channel. Luckily, since he lacked access to squid giant axons, he used the easily-obtained mixed nerve from the claw of the crab for this purpose. This proved to be a fortuitous choice since, in contrast to mammalian membrane fragments, these did not spontaneously reseal to form closed vesicles. This allowed substrates added to the... 

Mucoadhesive polymers exhibiting strong complexing properties are capable of inhibiting intestinal brush border membrane-bound proteases through a far distance inhibitory effect [65]. In vivo, the mucoadhesive polymer is separated from the brush border membrane by a mucus layer [30]. Although there is no direct contact between polymer- and membrane-bound enzymes, it could be shown that inhibition takes place. The exploitation of this far distance effect seems to be a very promising alternative to small molecular mass inhibitors, which are currently used as inhibitors of brush border membrane-bound proteases. 

The membrane-bound and purified enzymes lose little activity over a 2-3 month period when stored at room temperature in the presence of 4M NaCl. The purified ATPase is rapidly inactivated at 4°C and this is due to the dissociation of the enzyme [23]. The H. saccharovorum ATPase is more active in NaCl than in KCl, and most active when assayed between pH9 and 10 in the presence of at least 3.5M NaCl [19]. The membrane-bound enzyme rapidly loses activity when stored in 400 mM NaCl but is considerably more stable when either spermine (30 mM) or MgCl2 (100 mM) is present [34]. The purified ATPase is rapidly inactivated when stored in IM NaCl but is as stable in 500mM NaCl/lM (NH4)2S04 as in 4 M NaCl. The halobacterial ATPase is not inhibited by vanadate, azide, or Dio-9 [19] but is inhibited by DCCD when the enzyme is preincubated at pH 6 or less and at relatively high quantities of DCCD (125 nmol/mg protein). When membranes are incubated with C-DCCD, virtually all the radioactivity is associated with subunit II [19]. The conditions of inhibition are similar to those that inhibit F] activity [72-74]. This suggests that subunit II may be similar to the (3 subunit of F-type ATPases, an assumption consistent with the immunological reactivity of the subunit. Antiserum against the 3-subunit from the ATPase from 5. acidocaldarius which reacts with the 3 subunit from F-type ATPases [51] also reacts with subunit II from H. saccharovorum [60]. 

In the assay described here cells are permeablized using a detergent-based lysis method. Care must be taken to inhibit the membrane-bound enzyme, y glutamyl-transferase (y-GT), which metabolizes glutathione released from the cell (12). This is achieved by the use of an acidified lysis buffer (8). The lysate is then assayed for total protein and glutathione content. 

Studies of the degradation and regulation of oleamide revealed that it was hydrolyzed to oleic acid and ammonia by the action of a. membrane-bound enzyme, which based on the inhibition of its activity, seemed to be a serine or cysteine protease. Isolation and sequencing of the protein led to the cloning of its cDNA and expression in COS-7 cells. The expressed enzyme was found to not only hydrolyze oleamide but a number of other fatty acid amides and was thus designated as fatty acid amide hydrolase or FAAH (206). 

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