Membrane Protein Solubilization: Recent Advances and Challenges in Solubilization of Serotonin1A Receptors

Kalipatnapu, Shanti; Chattopadhyay, Amitabha

doi:10.1080/15216540500167237

Cited by 76 publications

(57 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The removal of the different types of lipids is based on their solubilization in organic solvents (chloroform, methanol, acetone, among others) in which they are soluble, followed by their separation/characterization by analytical methods such as thin layer chromatography (Prasad 1996). The removal of the proteins from the isolated membranes is the more toilsome process, because proteins tend to lose their structures if removed from the membrane into an aqueous medium (Yeagle 1993;Gennis 1989;Rigaud et al 1995;Le Maire et al 2000;White et al 2001;Magalhães et al 2003;Seddon et al 2004;Kalipatnapu and Chattopadhyay 2005). These laborious procedures may be needed for complex, multimeric, multi-domain proteins, not easily obtainable in recombinant form, e.g.…”

Section: Purification or Expression Of The Membrane Proteinmentioning

confidence: 99%

Proteoliposomes in nanobiotechnology

et al. 2012

View full text Add to dashboard Cite

Proteoliposomes are systems that mimic lipid membranes (liposomes) to which a protein has been incorporated or inserted. During the last decade, these systems have gained prominence as tools for biophysical studies on lipid-protein interactions as well as for their biotechnological applications. Proteoliposomes have a major advantage when compared with natural membrane systems, since they can be obtained with a smaller number of lipidic (and protein) components, facilitating the design and interpretation of certain experiments. However, they have the disadvantage of requiring methodological standardization for incorporation of each specific protein, and the need to verify that the reconstitution procedure has yielded the correct orientation of the protein in the proteoliposome system with recovery of its functional activity. In this review, we chose two proteins under study in our laboratory to exemplify the steps necessary for the standardization of the reconstitution of membrane proteins in liposome systems: (1) alkaline phosphatase, a protein with a glycosylphosphatidylinositol anchor, and (2) Na,K-ATPase, an integral membrane protein. In these examples, we focus on the production of the specific proteoliposomes, as well as on their biochemical and biophysical characterization, with emphasis on studies of lipid-protein interactions. We conclude the chapter by highlighting current prospects of this technology for biotechnological applications, including the construction of nanosensors and of a multiprotein nanovesicular biomimetic to study the processes of initiation of skeletal mineralization.

show abstract

Section: Purification or Expression Of The Membrane Proteinmentioning

confidence: 99%

Proteoliposomes in nanobiotechnology

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Dilute solutions of this surfactant (0.01-0.1% (w/v)) are often used in biotechnology during protein isolation to minimize protein-protein and protein-lipid interactions and to reduce the nonspecific bonding, therefore, to improve the final yield of the obtained proteins (24,25). With Triton X-100, we observed an increase in the pancreatin proteolytic activity in a concentration-dependent manner (Fig.…”

Section: Effect Of Non-ionic Surfactants On Pancreatin Proteolytic Acmentioning

confidence: 54%

Effect of Four Commonly Used Dissolution Media Surfactants on Pancreatin Proteolytic Activity

Guncheva

Stippler

2016

AAPS PharmSciTech

View full text Add to dashboard Cite

Abstract.Proteolytic enzymes are often used in dissolution testing of cross-linked gelatin capsules that do not conform to the dissolution specification. Their catalytic activity, however, can be affected when they are added to a dissolution media containing solubility enhancers, such as surfactants. The aim of this study was to assess the activity of pancreatic proteases in presence of four commonly used surfactants. We found that pancreatin exhibits remarkable proteolytic activity in the presence of Tween 80, even at the concentrations as high as 250 times its critical micelle concentration (cmc) in water, whereas, Triton X-100 enhanced the proteolytic activity of pancreatin when added at concentrations above its cmc in water. Both surfactants are non-ionic surfactants. On the other hand, sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), which are ionic surfactants, have a detrimental effect on the proteolytic activity of pancreatin. For example, a 50% reduction of the pancreatin activity was found in samples which contain a minor amount of SDS (0.05% w/v) in comparison to a surfactant-free reaction. Additionally, no activity was observed for the pancreatin-SDS samples which were incubated for 30 min at 40°C prior to testing. CTAB had an impact on pancreatin activity at concentrations higher than its cmc. Data from this manuscript can be used as a benchmark for optimization of the dissolution procedures that require use of both surfactants and enzymes.

show abstract

“…The latter observation appears to be consistent with the known effects of CHAPS on membrane structure. At low concentrations, detergent molecules tend only bind to the surface of the membrane, but as the CHAPS concentration increases, the membrane bilayer becomes disrupted (Kalipatnapu and Chattopadhyay, 2005). Such a model further explains why CHAPS disrupts the binding of the fully charged amitriptyline and chlorpromazine to HLM.…”

Section: Resultsmentioning

confidence: 83%

The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes

et al. 2015

View full text Add to dashboard Cite

Drugs and other chemicals frequently bind nonspecifically to the constituents of an in vitro incubation mixture, particularly the enzyme source [e.g., human liver microsomes (HLM)]. Correction for nonspecific binding (NSB) is essential for the accurate calculation of the kinetic parameters K m , Cl int , and K i . Many tyrosine kinase inhibitors (TKIs) are lipophilic organic bases that are nonionized at physiologic pH. Attempts to measure the NSB of several TKIs to HLM by equilibrium dialysis proved unsuccessful, presumably due to the limited aqueous solubility of these compounds. Thus, the addition of detergents to equilibrium dialysis samples was investigated as an approach to measure the NSB of TKIs. The binding of six validation set nonionized lipophilic bases (felodipine, isradipine, loratidine, midazolam, nifedipine, and pazopanib) to HLM (0.25 mg/ml) was shown to be unaffected by the addition of CHAPS (6 mM) to the dialysis medium. This approach was subsequently applied to measurement of the binding of axitinib, dabrafenib, erlotinib, gefitinib, ibrutinib, lapatinib, nilotinib, nintedanib, regorafenib, sorafenib, and trametinib to HLM (0.25 mg/ml). As with the validation set drugs, attainment of equilibrium was demonstrated in HLM-HLM and buffer-buffer control dialysis experiments. Values of the fraction unbound to HLM ranged from 0.14 (regorafenib and sorafenib) to 0.93 (nintedanib), and were generally consistent with the known physicochemical determinants of drug NSB. The extensive NSB of many TKIs to HLM underscores the importance of correction for TKI binding to HLM and, presumably, other enzyme sources present in in vitro incubation mixtures. IntroductionIn vitro approaches, using human liver microsomes (HLMs), human hepatocytes, or recombinant proteins as the enzyme source are used widely to characterize the kinetics of drug metabolism and drug-drug interaction potential (Houston, 1994;Obach, 1999;Rostami-Hodjegan and Tucker, 2007;Miners et al., 2010). However, numerous drugs, especially lipophilic organic bases and neutral compounds, bind nonspecifically to the microsomal membrane. Importantly, nonspecific binding (NSB) reduces the concentration of the unbound drug in the incubation medium, resulting in overestimation of the measured kinetic constants K m (or S 50 ) and the inhibitor constant K i . Consequently, in vitro intrinsic clearance (determined as V max /K m ) and inhibitory drug-drug interaction potential (assessed as [I]/K i , where [I] is the inhibitor concentration) are both underpredicted (Obach, 1999;McLure et al., 2000;Margolis and Obach, 2003;Grime and Riley, 2006;Miners et al., 2006Miners et al., , 2010. Thus, it is now widely accepted that NSB should be accounted for when kinetic parameters are calculated from in vitro metabolism and inhibition studies.Tyrosine kinase inhibitors (TKIs) are an emerging group of drugs used in the treatment of many forms of cancer and some other diseases (e.g., idiopathic pulmonary fibrosis). Typically, TKIs are lipophilic (log P values . 2....

show abstract

Membrane Protein Solubilization: Recent Advances and Challenges in Solubilization of Serotonin1A Receptors

Cited by 76 publications

References 64 publications

Proteoliposomes in nanobiotechnology

Proteoliposomes in nanobiotechnology

Effect of Four Commonly Used Dissolution Media Surfactants on Pancreatin Proteolytic Activity

The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes

Contact Info

Product

Resources

About