Membrane bound enzymes, solubilization

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. 

The membrane-bound enzymes participating in the assembly of the pep-tidoglycan carbohydrate chains were solubilized, and partially purified. 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 two heme groups have different oxidation-reduction potentials (Kroger and Innerhofer, 1976) one is the high-potential heme bp, the other the low-potential low-potential heme bp. For the membrane-bound enzyme, these potentials are Ey = —20 mV and m = —200 mV, respectively (Kroger and Innerhofer, 1976). For the detergent-solubilized QFR enzyme, the respective values are — 15 mV and — 150 mV (Lancaster et al., 2000). It has not yet been established which of the hemes bp and bp corresponds to bp and bp in W. succinogenes QFR. 

During cell fractionation, nearly all the cyclopenase activity was found in a fraction containing the cell wall together with the cytoplasmic membrane (83,84). From this fraction, the enzyme could be partially solubilized by detergents (e.g., Triton X-100) to yield a protein-phospholipid complex. By treatment with M-butanol, the solubilized enzyme preparation was split into the lipid fraction and the enzyme protein which retained a considerable part of the total enzyme activity. Compared with that of the membrane-bound enzyme, the substrate affinity of the solubilized protein-lipid complex was decreased, whereas... 

As indicated in Sections 1 and 2, succinate is an electron donor widely utilized for NAD(P) reduction by phototrophic purple bacteria. The membrane-bound enzyme responsible for succinate oxidation has been solubilized and partially characterized in the purple non-sulfur bacteria R. rubrum [73,74] and Rhodopseudo-monas sphaeroides (recently renamed Rhodobacter sphaeroides) [57]. In situ characterization of the iron-sulfur centers likely to be associated with succinate dehydrogenase has been accomplished for Rps. capsulata [59] and C. vinosum [51]. Of particular interest is the presence of a succinate-reducible [51,57,58,73] and fu-marate-oxidizable [51] iron-sulfur cluster with near +50 mV that, like center S-3 [60,61,75,76] of mitochondrial succinic dehydrogenase (Complex II), is paramagnetic in the oxidized state. The enzyme in phototrophic bacteria also appears to have one or two ferredoxin-like (i.e., paramagnetic in the reduced state) iron-sulfur centers that correspond to centers S-1 (succinate-reducible, EJ ranging from... 

M LiCl[29]. Several of the membrane-bound enzymes in Sulfolobus are loosely bound and are solubilized following high pressure decompression [2,30]. 

Once soluble, the membrane-bound enzymes are much like cytoplasmic enzymes and can be purified using similar methods. Excellent descriptions of such methods can be found in ref. [36]. These methods are only limited by circumstances such as the salt dependence of the proteins from extreme halophiles and the cold-sensitivity exhibited by some membrane-bound enzymes when solubilized. 

The mechanism of release of membrane-bound enzymes such as GGT and ALP into the circulation is less well understood. There appears to be increased synthesis of GGT and ALP in diseased human liver. How this enhanced synthesis of tissue-bound enzymes translates into increased activity in plasma is not clear. However, fragments of hepatocyte niembrane rich in GGT and ALP activity have been detected in plasma of patients with cholestasis, a process that may be a result of membrane fragmentation by bile acids. Furthermore, bile acids, which are detergents, could solubilize and release GGT and ALP from plasma membranes. In vitro studies of membranes treated with bile acids demonstrate that this possibility exists. ... 

S-Adenosyl-L-methionine ll-Methoxy-2,16-dihydro-16-hydroxytaberso-nine A-methyltransferase (NMT, EC 2.1.1.99) transfers a methyl group from S-adenosyl-L-methionine to N-1 of ll-methoxy-2,16-dihydro-16-hydroxytabersonine. The enzyme has a high substrate specificity, the reduced 2,16 double bond in the tabersonine skeleton being essential (242). Absence of the 16-hydroxy group resulted in 60% lower A-methylation rate, compared with the natural substrate. Tabersonine is not accepted as substrate. Also, the 14,15-double bond is an essential requirement for acceptance as substrate. The enzyme is localized in the thylakoids of chloro-plasts (181). The partially purified enzyme was further characterized (243). It could be solubilized with CHAPS. By sucrose gradient centrifugation an apparent Mr of 60,000 was found. The solubilized enzyme showed some differences in substrate specificity compared with the membrane bound enzyme. 

Aliphatic alcohols such as ethanol, n-propanol, n-butanol and isopentyl (isoamyl) alcohol exert disruptive effects upon bacterial membranes as shown by the lysis of protoplasts [76]. Of these, only ethanol displays any useful bactericidal activity it is used to cleanse the skin before injections and to sterilize instruments. Butanol is used to solubilize membrane-bound enzymes [22,91], its action being similar to that of the non-ionic detergents described below. 

Membrane-bound enzymes, as several authors have emphasized, are generally bound to lipid and resistant to solubilization unless the lipid is removed or lipid-protein interactions reduced. The forces between the lipid and protein elements are unlikely to be the same for all enzymes nor the same for the same enzymes from different species. Lipid removal by surfactants results in the exchange of bound lipid for bound detergent molecules [44] whether or not this occurs without change in the conformation of the protein is open to question. Siekewitz [91] has pointed out other problems in interpreting results of solubilized proteins ... 

The vacuoles in higher plants are essential for the maintenance and regulation of the homeostatic environment of the cells [1]. The vacuolar membranes of plant cells contains an electrogenic H -ATPase which provides the proton-motive force for the active transport of solutes across the vacuolar membranes. The activities of certain membrane-bound enzymes are lost by the removal of constituted lipids, but are restored after the addition of exogenous phospholipids [2,3]. Kasamo [4] showed that the various molecular species of PC activated to different extents the H -ATPase solubilized from the plasma membrane of rice cultured cells. 

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. ... 

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