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Biomedical subjects

H Bayley

Publications and source records attributed to H Bayley.

102 records · Page 6Linked to original sources
PubMed1981

Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments.

The complete denaturation and subsequent renaturation and reconstitution of a polytopic integral membrane protein are demonstrated. Delipidated bacteriorhodopsin (Huang, K.-S., Bayley, H., and Khorana, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 323-327) is completely denatured when transferred into 88% formic acid or anhydrous trifluoroacetic acid as shown by NMR and circular dichroism spectroscopy. When ethanol is added to a solution of the denatured protein, helical structure is largely reformed. After neutralization of the acid with ammonia and dialysis against a solution of sodium dodecyl sulfate a substantial amount of this structure is retained. Complete renaturation, characterized by the formation of the chromophore, occurs when phospholipids, cholate, and retinal are added to the sodium dodecyl sulfate solution of the protein. After dialysis of the solution to remove the detergents, the bacteriorhodopsin assembles into vesicles that are fully active in light-driven proton translocation. We also show that two chymotryptic fragments of bacteriorhodopsin (residues 1-71 and 72-248), separated under denaturing conditions, can be made to reassociate and form active vesicles with phospholipids.

Apoproteins↗
PubMed1981

Site of attachment of retinal in bacteriorhodopsin.

The identity of the lysine residue in bacteriorhodopsin to which the chromophore, retinal, is attached as a Schiff's base has been reinvestigated. Retinal is now shown to be linked to lysine-216 and not lysine-41, as had been concluded previously. The retinal in purple membrane was replaced by [15-3H]retinal, the Schiff's base linkage was reduced with NaBH4 while the sample was illuminated, and the resulting [retinyl-3H]bacterio-opsin was cleaved with CNBr. The radioactivity was present exclusively in the COOH-terminal peptide (amino acids 210--248). Sequence analysis showed that the [3H]retinal was attached to lysine-216. The same site was labeled when purple membrane was reduced with NaBH4 in the light at pH greater than 10 or at pH 8 and when membranes modified with ether or solubilized with hexadecyltrimethylammonium bromide were reduced in the dark. Our finding places of Schiff's base linkage close to the midpoint of the putative membrane-spanning alpha-helix that is directly connected to the COOH terminus of bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1980

Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane.

1-[3H]Spiro[adamantane-4,4'-diazirine], a lipophilic, photoactivatable reagent designed to label those segments of intrinsic proteins that lie within the lipid bilayer of biological membranes, has been evaluated. The reagent labels the intrinsic proteins of human erythrocyte membranes far more heavily than it labels the extrinsic proteins. This result, together with a detailed analysis of the label distribution in several well-characterized membrane proteins [Goldman, D.W., Pober, J.S., White, J., & Bayley, H. (1979) Nature (London) 280, 841], demonstrates that labeling with adamantanediazirine is a convenient and rapid method both for distinguishing intrinsic from extrinsic membrane proteins and for locating within intrinsic proteins those amino acid residues that are in contact with the hydrocarbon core of the lipid bilayer.

Adamantane↗
PubMed1980

Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid.

Solubilizations of the purple membrane from Halobacterium halobium with the detergent Tritain X-100 followed by gel filtration in deoxycholate solution gave bacteriorhodopsin that was more than 99% free from endogenous lipid. The delipidated bacteriorhodopsin was reconstituted with exogenous phospholipids to form vesicles which on illumination efficiently translocated protons. The direction of proton pumping was from the outside to the interior of the vesicles, indicating that the orientation of bacteriorhodopsin in the vesicles was opposite to that in the bacterial membrane. This orientation was confirmed by cleavage of the carboxyl terminus of the protein by proteolysis from the outside of the vesicles.

Bacteriorhodopsins↗
PubMed1978

Photogenerated reagents for membrane labeling. 1. Phenylnitrene formed within the lipid bilayer.

Phenylnitrene generated photochemically from phenyl azide that is bound to artificial phospholipid vesicles labels the fatty acid chains of the lipids in low yield. The labeling yield varies from approximately 3.3% with soybean lecithin (which is highly unsaturated) to approximately 0.25% with dimyristoyllecithin (which is completely saturated). Labeling is largely eliminated by reduced glutathione in the aqueous phase. Nitrenes are evidently unsatisfactory reagents for the labeling either of lipids or by analogy of the hydrophobic portions of membrane proteins. This is mainly because the long lifetimes and electrophilic character of nitrenes will lead to the preferential labeling of extrinsic membrane components. Phenyl azide itself is further compromised as a lipophilic reagent by its rather low partition coefficient into lipid bilayers, as measured by equilibrium dialysis.

Azides↗
PubMed1978

Photogenerated reagents for membrane labeling. 2. Phenylcarbene and adamantylidene formed within the lipid bilayer.

Phenylcarbene and adamantylidene have been generated photochemically from the corresponding diazirines within lipid bilayers. Reasonable yields of labeled fatty acid side chains have been observed. The products have been characterized by gas chromatography-mass spectrometry and derive both from the insertion of the carbene into carbon-hydrogen bonds of saturated fatty acids and from the addition of the carbene to the carbon-carbon double bonds of unsaturated fatty acids. In contrast to the results found using phenylnitrene, the lipid labeling by carbene is not reduced by the water-soluble scavenger glutath ione. Carbenes generated from diazirines are evidently superior reagents for the photolabeling of lipids and should be useful for identifying the intrinsic hydrophobic sections of membrane proteins.

Adamantane↗
PubMedDate not supplied

A molecular mechanism for long-term sensitization in Aplysia.

Sensitization of the gill- and siphon-withdrawal reflex in Aplysia is thought to result from a set of molecular processes with different time courses: short-term sensitization is explained by cyclic AMP-dependent modulation of ion-channel function in sensory neurons lasting minutes; memory that endures for hours or longer, by the expression and distribution within the neurons of new gene products. Because gene induction and axonal transport are relatively slow, there may also be a need for a distinct form of intermediate memory to bridge the short- and long-term processes. We now report that a protocol producing long-term sensitization results in a decrease in the amount of regulatory subunits of the cAMP-dependent protein kinase in animals 24 h after training, with no effect on the catalytic subunit. The loss appears to be post-translational. Because a decrease in the ratio of regulatory to catalytic subunits would result in elevated kinase activity after cAMP has returned to its unstimulated concentration in sensory cells, it could be the molecular mechanism of intermediate memory.

Animals↗