Purification and reconstitution

Transhydrogenase was also purified somewhat later and independently by a more complicated procedure which, however, also produced the 115000 molecular weight polypeptide [102]. Subsequently, additional improved methods have been published, which involve the use of immobilized antibodies [103] and affinity chromatography on immobilized NAD [104] or NADP [105]. With all preparations reconstituted transhydrogenase was shown to be a proton pump by both an indirect assay using 9-aminoacridine as pH probe for the interior space of the vesicles [106], and a direct assay using a pH electrode [107]. 

Most of the procedures published so far have been either unsuitable for larger scale preparations (maximally 1 mg) or irreproducible. In an attempt to overcome these problems a purification procedure has been developed recently [108] which is a modification of a previous method [83]. The modified procedure involves FPLC (fast protein liquid chromatography), is rapid, reproducible and allows the preparation of large amounts of active enzyme. Purified transhydrogenase obtained by this procedure is reconstitutively active and pumps protons as judged by quenching of 

Comparison of methods for the purification of mitochondrial nicotinamide nucleotide transhydrogenase from beef heart 

Unless otherwise indicated transhydrogenase activity was assayed by reduction of AcPyAD by NADPH and protein was determined by the Lowry method 

Preparation Spec. act. (fimol min mg ) Purification factor Yield (%) Transhydro- genase (mg/prep) Proton pumping 

---

Ruan Z-S, V Anantharam, IT Crawford, SV Ambudkar, SY Rhee, MY Allison, PC Maloney (1992) Identification, purification, and reconstitution of OxlT, the oxalate formate antiport protein of Oxalobacter formigenes. J Biol Chem 267 10537-10543. 

GILLAM, E.M.J., BABA, T, KIM, B-R., OHMORI, S., GUENGERICH, F.P., Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme, Arch. Biochem. Biophys., 1993, 305, 123-131. 

Danbolt, N. C., Pines, G., and Kanner, B. I. (1990) Purification and reconstitution of the sodium- and potassium-coupled glutamate transport glycoprotein from rat brain. Biochemistry 29,6734-6740. 

Das A, Ivey DM, Ljungdahl LG. 1997. Purification and reconstitution into prote-ohposomes of the FiFq ATP synthase from obligately anaerobic bacterium Clostridium thermoautotrophicum. J Bacteriol 179 1714—20. 

Human CYP1A2 has been expressed in several microbial systems including bacteria and yeast (Sandhu et al., 1994 Sengstag et al., 1994 Guo et al., 1994). As with human CYPlAl, the turnover numbers for E. co/Z-expressed human CYP1A2 are higher upon purification and reconstitution. 

Multiplicity of Cytochrome P450, Purification, and Reconstitution of Cytochrome P450 Activity. Even before appreciable purification of CYP had been accomplished, it was apparent from indirect evidence that mammalian liver cells contained more than one CYP enzyme. Subsequent direct evidence on the multiplicity of CYPs included the separation and purification of CYP isozymes, distinguished from each other by chromatographic behavior, immunologic specificity, and/or substrate specificity after reconstitution and separation of distinct polypeptides by sodium... 

As discussed above, the purification and reconstitution of active PKSs from a variety of heterologous expression systems (including E. coli) are now feasible. Given the substantial tolerance of PKSs toward altered substrates and intermediates, it should therefore be possible to exploit this catalytic potential in a far more powerful way in cell-free systems than in intracellular systems. The primary limitations are with regard to the scale of synthesis. Attempts to stabilize and reuse the enzymes, in conjunction with the development of cheaper sources of natural and unnatural substrates and recycling systems for NADPH, should go a long way toward ameliorating this limitation. 

Koepsell, H. (1986). Methodological aspects of purification and reconstitution of transport proteins from mammalian plasma membranes. Rev. Physiol. Pharmacol. 104,65-137. 

Koepsell, H Menuhr, H., Ducis, I., Wissmuller, T.F. (1983). Partial purification and reconstitution of the Na+- D-glucose cotransport protein from pig renal proximal tubules. J. Biol. Chem. 258, 1888-1894. 

KOCHS, G., GRISEBACH, H., Phytoalexin synthesis in soybean purification and reconstitution of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase. Arch Biochem. Biophys., 1989,273,543-553. 

Ambudkar SV, Lelong IH, Zhang J, Cardarelli CO, Gottesman MM, Pastan I (1992) Partial purification and reconstitution of the human multidrug-resistance pump characterization of the drug-stimulatable ATP hydrolysis. Proc Natl Acad Sci USA 89 8472-8476... 

These data along with earlier studies on a TrpE-Stel4p fusion protein show that Icmt is the sole enzymatic component necessary to methyl ester-ify the a-carboxyl of isoprenylated cysteine residues [43,49]. Unfortunately, despite significant efforts, hicmt has proven refractory to functional purification and reconstitution. Active hicmt could not be extracted with detergent from leukocyte membranes in active form nor could its enzymatic activity be restored upon reconstitution into liposomes [23]. 

These conclusions have been drawn from whole-cell studies. Evidently, conclusive evidence for primary Na translocation coupled to CO2 reduction to formyl-MFR will require the purification and reconstitution of the components involved and demonstration of a direct role of Na in the reactions catalyzed by the reconstituted system. 

Much effort has been put into the isolation, purification and reconstitution of both photosystems. Work on PSII has been concerned especially with the nature of the dioxygen-evolving site, which is thought to be a manganese protein. ESR studies on spinach chloroplasts have led to the postulate of the involvement in oxygen evolution of a pair (or possibly a tetramer) of antiferromagnetically... 

U Pick and E Packer (1979) Purification and reconstitution of the N,N -dicyclohexylcarbodiimide-sensitive AT-Pase complex from spinach chloroplasts. J Biol Chem 254 2793-2799... 

E Schneider and K Altendorf (1987) Bacterial adenosine 5 -triphosphate synthase FiFq) purification and reconstitution of Fq complexes and biochemical and functional characerization of their subunits. Microbiol Rev 51 477-497... 

Purification and reconstitution of the enzyme system will be required before elucidation of the mechanism of its activation. 

Much of what is known about these protein-lipid interactions has come from protein purification and reconstitution of function dependent on lipids. Genetic approaches coupled with in vitro verification of function have uncovered new roles for lipids. Most exciting have been results from X-ray crystallographic analysis of membrane proteins, which have revealed lipids in specific and tight association with proteins. The predominant structural motif for the membrane-spanning domain of membrane proteins is an a-helix of 20-25 amino acids, which is sufficient to span the 30-A core of the bilayer. A 3-barrel motif is also found to a lesser extent. 

L.W. Parks, and D.E. Kelly (1995). Purification and reconstitution of activity of Saccharomyces cere-visiae P450 61, a sterol 22-desaturase. FEES Lett. 377, 217-220. 

Jenkins, G.H. Subrahmanyam, G. Anderson, R.A. Purification and reconstitution of phosphatidylinositol 4-kinase from human erythrocytes. Biochim. Biophys. Acta, 1080, 11-18 (1991)... 

The transport speed of the transporter is equivalent to the ion flux of the ion channel. However, the ion flux of channels does not exhibit saturation with an increase of the ion concentration and it is larger by orders of magnitude than the transport speed of transporters. The ion flux is directly proportional to the ion stream. The basis of the function of translocators is membrane-enclosed compartments cells, organelles, vesicles, or proteoliposomes (prolis). Prolis and liposomes play a role in purification and reconstitution of translocators. The following sections are dedicated to them. 

Barzilai, A., et al. (1984). Isolation, Purification and Reconstitution of the Na Gradient-dependent Ca Transporter (Na -Ca Exchanger) from Brain Synaptic Plasma Membranes, Proc. Natl Acad. Sci. USA 81 6521-6525. 

Characterization of membrane proteins in isolated native cellular membranes by dynamic nuclear polarization solid-state nmr spectroscopy without purification and reconstitution was carried out by Jacso et alP ... 

---