Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Application to subcellular fractions of the adrenal medulla

Pryde, James G.; Phillips, John H.

doi:10.1042/bj2330525

Cited by 149 publications

(79 citation statements)

References 36 publications

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“…Alternatively the mannosidase may be a peripheral membrane protein or an integral membrane protein with pronounced hydrophilic character. For example, heavily glycosylated proteins with a single transmembrane domain are often recovered in the aqueous phase of Triton X-214, due to self-association through the hydrophobic domains or detergent binding to these domains [33,34]. Similar behaviour has also been noted for integral membrane proteins such as Thy1 antigen, which are anchored to the membrane through an amphipathic phosphatidylinositol tail 1351.…”

Section: Discussionmentioning

confidence: 78%

Purification and characterization of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase from rat liver

Bonay

Hughes

1991

European Journal of Biochemistry

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We have identified a mannosidase in rat liver that releases a1 + 2, a1 + 3 and a1 + 6 linked manose residues from oligosaccharide substrates, Man,GlcNAc where n = 4-9. The end product of the reaction is Manal + 3[Manal --f G]ManPl+ 4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a K, of 65 pM and 110 pM for the Man,GlcNAcoligosaccharide or Man,GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fez+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the a1 + 2 linked residue followed by hydrolysis of a1 + 3 and a1 + 6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-amannoside nor the hybrid GlcNAc Man,GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50 -500-fold higher than required for complete inhibition of Golgimannosidase I1 and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases.The major pathway of N-glycan biosynthesis involves removal of three glucose residues from the asparagine-linked Glc3Man9GlcNAc2 oligosaccharide, by glucosidases I and I1 [I] and up to four a1 + 2 linked mannose residues by distinct mannosidases of which several have been purified and well characterized [2 -151 such as endoplasmic reticular mannosidase and Golgi mannosidases IA/IB. The smallest oligomannose structure resulting from the concerted action of these enzymes is Man,GlcNAc2Asn, the preferred substrate for GlcNAc transferase I. The product of this reaction, GlcNAcMan,GlcnAc, Asn is then hydrolysed by Golgi mannosidase 11, releasing an a1 + 3 and an a1 + 6 linked mannose residue yielding a GlcNAcMan3GlcNAc2 intermediate which is modified further by subsequent addition of GlcNAc residues and branch elongation [I].Previously, we reported evidence for the presence in crude BHK cell extracts, of a mannosidase able to release a1 + 3 and a1 + 6 linked mannose from a Man5GlcNAc oligosaccharide leading to the formation of the tetrasaccharide Manal + 3[Manal + 6]Manp1 +4GlcNAc [16]. By various criteria, the activity was shown to be different from other a-mannosidases previously described. However, due to the fact that there is no information on purified a-mannosidases from BHK cells, the possibility could not be excluded that the Man,GlcNAc mannosidase was the BHK cell homologue of a well-characterised processing mannosidase, perhaps with a

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Section: Discussionmentioning

confidence: 78%

Purification and characterization of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase from rat liver

Bonay

Hughes

1991

European Journal of Biochemistry

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“…Although this partitioning is not a solubilization protocol per se (Pryde, 1986), it allows an approximate determination of the hydrophobicity of a protein and has been successfully used in animal (Pryde and Phillips, 1986) and plant systems. A recent example is the resolution of the membrane localization of the fusicoccin receptor in Commelina communis tissues (Oecking et al, 1994).…”

Section: Hydrophobic Phase During Triton X-114 Partitioningmentioning

confidence: 99%

“…The published method (Pryde and Phillips, 1986) was followed exactly with one exception: the aqueous phase obtained after the SUC cushion separation was used directly without dialysis. As a consequence, a small amount of contamination by the detergent phase may be present.…”

Section: Triton X-114 Phase Partitioningmentioning

confidence: 99%

The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein

Bernasconi¹,

Patel²,

Reagan³

et al. 1996

Plant Physiology

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N-1 -Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbifa pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes.

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“…Triton X-114 was obtained from Fluka AG and purified by three cycles of solution and condensation [17]. The concentration of the final stock solution of Triton X-114 was measured as described [18].…”

Section: Methodsmentioning

confidence: 99%

Allosteric Regulation of Proton Translocation by a Vacuolar Adenosinetriphosphatase

Webster¹,

Perez-Castiñeira²,

Atkins³

et al. 1995

Eur J Biochem

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The kinetics of nucleoside-triphosphate-dependent proton translocation by a vacuolar-type adenosinetriphosphatase have been studied, using the enzyme from bovine chromaffin-granule membranes, purified and reconstituted into proteoliposomes. The reaction was followed by recording the quenching of the fluorescence of the permeant weak base 9-amino-6-chloro-2-methoxyacridine; fluorescence data were collected and stored in digital form, and the initial reaction rates estimated by linear regression. In the absence of nucleoside diphosphate, the dependence of initial rates of proton translocation on substrate concentration were fitted well by the Michaelis-Menten equation, as were the kinetics of ATP hydrolysis.ADP and other nucleoside diphosphates were potent inhibitors of the ATPase, effecting a reduction in the maximum velocity of the reaction, and producing sigmoid substrate-saturation curves which could be fitted by the empirical Hill equation, the Hill coefficient approaching 2 at high inhibitor concentrations. Data sets containing initial-rate estimates were collected over a wide range of independently varied concentrations of substrate and inhibitor and were modeled, using rate equations derived from several different models based on the concerted-transition model of allosteric inhibition proposed by Monod, Wyman and Changeux. These equations were fitted to the data by weighted non-linear regression, using an iterative computer program to obtain the best estimates of kinetic parameters. One model consistently fitted all of the data sets better than all the others, and this model was based on the following assumptions:that the ATPase exists in two conformational states, R and T ; that only the R state is catalytically active; that each state contains three kinetically equivalent catalytic sites, and one regulatory site; that nucleoside triphosphates bind only to the catalytic sites, and that nucleoside diphosphates bind both to the catalytic sites and to the regulatory site. The optimized values of the kinetic parameters indicate that in the absence of nucleoside diphosphate, the enzyme is almost completely in the R state ; that nucleoside triphosphates bind more tightly to the R than to the T state; that binding of nucleoside diphosphates to the regulatory site is very tight, but occurs only in the T state; and that competitive binding of nucleoside diphosphates at the catalytic sites is stronger in the T state than in the R state. Experiments conducted with varying total magnesium concentrations indicated that the magnesium complexes of nucleoside diphosphates are much stronger inhibitors than the free nucleotides, and that free nucleoside triphosphates are weakly inhibitory, probably competing with the magnesium complexes for binding at the catalytic sites. The results of these experiments indicate that the effects of nucleoside diphosphates, particularly ADP, occur at concentration ranges that are likely to be physiologically significant, and they make predictions about ligand-induced conformation changes i...

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Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Application to subcellular fractions of the adrenal medulla

Cited by 149 publications

References 36 publications

Purification and characterization of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase from rat liver

Purification and characterization of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase from rat liver

The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein

Allosteric Regulation of Proton Translocation by a Vacuolar Adenosinetriphosphatase

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