Membrane-bound enzymes isolation

Purified CF solubilized from chloroplast membranes has no ATPase activity, unless activated by trypsin, heat or dithiothreitol but like the membrane-bound enzyme, isolated CF can bind nucleotides in a non-covalent and nonenergy-requiring process. The maximum number of bound nucleotides varies, depending on the way the complex AdN-CF is recovered from the excess of free nucleotides. A conflicting point is the specificity of these sites for ATP or ADP and the effect of Mg on the binding or exchange of nucleotides on CF. ... 

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. 

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... 

PBPs are membrane-bound enzymes. In general, PBPs, especially high-molecular-mass PBPs, consist of a short cytoplasmic domain in their N-terminus, followed by a hydrophobic transmembrane domain, then a transglycosylase domain (for class A PBPs) or a non-penicillin-binding domain (also called an unknown functional domain for class B PBPs), and finally a transpeptidase domain located in their C-terminus (Fig. 1 [1,2,15,56]). Thus, purification of PBPs traditionally involved the isolation of membranes and solubilization of PBPs from the membrane preparation by detergents. Since PBPs are present at a very low level (approximately 100-300 molecules per cell) [57-59] and also in multiple forms in the cell, this classical approach was not particularly efficient for producing a significant amount of a purified PBP protein of particular interest. 

The use of artificial lipid membranes and isolated membrane fragments and vesicles has been of great value in the study of membrane function, particularly transport and membrane-bound enzyme reactions. Current research in this area uses sophisticated techniques for determining the molec-... 

It may appear difficult to reconcile the differences in data from the two Russian groups. But one possible explanation is that Mansurova et al. [71,74] have extracted only part of PPase II, possibly subunits a and )8. The isolation method they actually have used is somewhat undetailed, but if it only consists of a buffer extraction from an acetone powder of mitochondria, without added detergents, one may well envisage that only part of the membrane-bound enzyme is liberated. The same may be true for their sucrose washing of submitochondrial particles with or without sonication. 

The redox-active natural product (+)-methanophenazine (MP) is the first phenazine to be isolated from archea. This compound is able to mediate the electron transport between membrane-bound enzymes and was characterized as the first phenazine derivative involved in the electron transport of biological systems. The research team of U. Beifuss prepared this natural product by using the Williamson ether synthesis in the last step of the synthetic sequence. The etherification was conducted under phase-transfer conditions in a THF/water system in the presence of methyltrioctyl-ammonium chloride and using potassium hydroxide as a base. 

The description given above shows the potentiality of the method and suggests that other membrane-bound enzymes could be isolated by this procedure. 

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). 

The reduction of RA to VA is catalyzed by cis-9, tram-11 octadecadienoic acid reductase. Isolation of this membrane-bound enzyme from B. fibrisol-vens indicates that it has an absolute requirement for iron and expresses maximal activity at a pH between 7.2 and 8.2 (Hughes et al., 1982). Pure culture studies with B. fibrisolvens also have shown that the reductase is not... 

These nitrite reductases are described as monomers of about 60 kDa, containing six c-type hemes, five being low-spin and one high-spin in the as-isolated state [134]. However, recent evidence has indicated that the situation maybe more complex. In E. coli, the gene for the nitrite reductase (nrfA) codes for a 50 kDa cytochrome containing four (or five), rather than six heme-binding sites [135]. The membrane-bound enzymes were shown not to be monomers,... 

A. thaliana mutants have also provided a means of eloning genes difficult to isolate by other methods. As in other kingdoms, the membrane-bound enzymes of plants have been... 

Ferredoxin-NADP oxidoreductase is the final enzyme of the photosynthetic electron transport chain (for a review see Ref. 1). This fla-voprotein was isolated from spinach and lettuce chloroplasts closely associated with a trimer of a 17.5 kDa polypeptide (2,3). The purified complex showed some of the allotopic properties of the membrane-bound enzyme like pH and temperature profiles of diaphorase activity (2,4,5). Immunological evidence indicates that the binding protein protrudes from the thylakoids into the stroma (6) and that it is affected by the extent of thylakoids energization, suggesting that it may be the regulatory subunit of the reductase. 

The clear detection of both reversible active-site and biocatalytic waves represents a completeness that establishes the feasibility of applying direct elec-tochemistry to probe the mechanism of action of complex redox enzymes. To take this further, I shall take up the author s prerogative for mentioning studies currently underway in the laboratory and mention, briefly, another membrane-bound enzyme, fumarate reductase (FR), isolated from Escherichia coll Structurally, this is closely related to the more familiar succinate dehydrogenase (SDH) which constitutes the major part of Complex II of the mitochondrial respiratory chain. Of the four subunits which make up the membrane-bound system, two may be freed to give a soluble enzyme that is active in fumarate reduction by artificial electron donors [230]. The larger of these, MW approx. 70000, contains, like SDH, a covalently bound FAD. The smaller, MW approx. 30000, appears to contain three Fe-S clusters. These are termed centre 1 ([2Fe-2S]), centre 2 ([4Fe-4S]) and centre 3 ([3Fe-4S]). Their respective reduction potentials as determined by potentiometry are — 20 mV, — 320 mV, and — 70 mV [231], (Although the potential of the FAD has not been determined for FR, the two-electron value for beef heart SDH is — 79 mV at pH 7.0. The radical form is unstable since the two one-electron reductions occur at potentials of — 127 and — 31 mV respectively [232].)... 

Purification of p-o-galactosidase Isolation of the apo-form of ovoflavoprotein Isolation of P-lactamases Purification of a cysteine-containing histone Separation of the principal glycoproteins in human red-cell membranes from other proteins Purification of corticosteroid-binding globulin Purification of transcortin Separation of membrane-bound enzymes Studies of the effect of the chain-length on the adsorption 125 of albumin removal of albumin from sera... 

Lipids play an essential role in the structure and function of biological membranes. The concept that the lipid enviroment influences the activity of membrane bound enzymes is now generally accepted. The sarcoplasmic reticulum(SR) of skeletal muscle is an intracellular membranous system with an important physiological role in contraction and relaxation (Hasselbach, 1964 Martonosi, 1971). The Ca2+ transport and (Ca2++ Mg2+)-ATPase activities which exhibit isolated SR preparations have been shown to be dependent on membrane phospholipids (Martonosi, 1968). An involvement of the fatty acid component of phospholipids in the functioning of the Ca2+ pump has also been suggested (Seiler et al., 1970 Seiler Hasselbach,... 

Besides stimulating the production and release of certain hydrolases, GA can also retard enzyme activity. Consistent with the observation that GA induces degradation of aleurone cell walls is the observation that GA inhibits the synthesis of pentosan (arabinose and xylose) components of the wall. Between 4 and 16 h after GA addition to isolated aleurone tissue there is a marked decline in pentosan biosynthesis, in part due to the reduced activity of a membrane-bound arabinosyl transferase, and perhaps also of xylosyl transferase [62]. Changes in such membrane-bound enzymes could reflect a shift in the role of membranes from the production of cell wall components to the production, or packaging of hydrolases. 

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