Binding of lithium diiodosalicylate to glycophorin.

Glycophorin was purified from human erythrocyte ghosts by the lithium diiodosalicylate -phenol procedure utilizing 125I-labeled lithium diiodosalicylate. The glycophorin preparation was found to contain 8.9 +/- 2.1 mol lithium diiodosalicylate per mol glycophorin. This bound lithium diiodosalicylate cannot be removed by extensive washings with a variety of polar organic solvents nor by treatment with the detergent, sodium deoxycholate. Further, the hydrophobic peptide produced from glycophorin by trypsin digestion contained 3.4 mol lithium diiodosalicylate per mol peptide.

Detergent removal during membrane reconstitution.

Efficiency of detergent removal during the course of several different procedures for membrane protein reconstitution was examined. Reconstitution methods studied include ethanol injection-dialysis, detergent dialysis and detergent-gel filtration. In the ethanol injection-dialysis method, approx. 70 molecules of ethanol per 1000 molecules of phospholipid are retained even after extensive (150 h) dialysis. Efficiency of detergent removal by dialysis depends on the detergent. However, even for sodium deoxycholate, a detergent possessing a large critical micelle concentration, there are approx. 7 molecules of deoxycholate per 1000 molecules of phospholipid retained by the bilayer even after extensive (310 h) dialysis. Detergent removal by gel filtration (Sephadex G-200 or G-50) of deoxycholate, cholic acid and Triton X-100 is more efficient than removal by dialysis; as few as 10 molecules of deoxycholate are retained per 100 molecules of phospholipid after one column passage, taking only a few hours. Ethanol was less efficiently removed by one passage over a Sephadex column than by extensive dialysis. Removal of Triton X-100 by passage over, or dialysis against, Biobeads SM-2 resulted in a similar level of detergent retention to that found by passage over Sephadex G-200 or G-50. Utilizing gel-filtration techniques, we have examined the competition for the hydrophobic peptide of glycophorin, T(is), between sodium deoxycholate and a series of phospholipids as a possible means of obtaining a quantitative measure of protein-lipid affinity. On the basis of these preliminary studies we conclude that the T(is) peptide has a relative lipid affinity of phosphatidylinositol > phosphatidylcholine > phosphatidylserine.

Laser Raman spectra of oxidized hydroperoxidases.

Resonance Raman spectra of oxidized hydroperoxidases are examined for shifts in the structure-sensitive, anomalously polarized bands; these are found, respectively, at 1576, 1567 and 1570 cm-1 in the high-spin resting enzymes: horse radish peroxidase, horse blood catalase, and cytochrome c peroxidase. In compound II of horse radish peroxidase and horse blood catalase, and in the enzyme-substrate complex of cytochrome c peroxidase, this band appears at 1587-1590 cm-1 and indicates the iron atom is now in-plane with the porphyrin ring. Weak Raman scattering found with horse radish peroxidase I is consistant with a porphyrin eta-cation radical formulation.

The glycophorin-phospholipid interface in recombined systems. A 31P-nuclear magnetic resonance study.

Glycophorin, the MN glycoprotein from the erythrocyte membrane, was recombined with egg phosphatidylcholine and with the total lipid extract from human erythrocyte membranes in a membranous form. 31P-nuclear magnetic resonance (NMR) spectra of the recombinants resembled spectra obtained from unsonicated phospholipid dispersions and biological membranes. The glycophorin/phospholipid ratio in these recombinants was varied from approximately 50:1 (lipid/protein) to 200:1, and 31P-NMR spectral intensities were obtained. Comparison of these intensities to that expected based on a pure phospholipid standard revealed that there were two phospholipid environments in the recombinants: one immobilized by the protein, and one slightly disordered and nonimmobilized. A relatively constant number of phospholipids were immobilized per glycophorin at all lipid/protein ratios studied.

Interaction of the lipid-associating domain of glycophorin with phospholipid bilayers.

The MN-blood group bearing glycoprotein of the human red cell membrane, glycophorin, has been shown to traverse the membrane in vivo such that the NH2-terminal portion is exposed to the external, and the COOH-terminal portion exposed to the cytoplasmic, mileiu. The intervening 23 residue lipid-associating domain (LAD) of glycophorin has been shown to have a unique overall hydrophobicity. The LAD of glycophorin can be obtained intact within an aqueous insoluble tryptic peptic, T(is). Under appropriate conditions (the reaction not being spontaneous), T(is) can be associated with phospholipid bilayers. Freeze fracture studies of T(is): phospholipid vesicles suggest that T(is) forms multimeric torus-shaped intramembranous structures 80 A in diameter with n greater than 4. The plot of T(is) concentration versus multimer density suggests there is a critical multimer concentration (CMC) for T(is) in phospholipid bilayers (L/P = 200/1). Various physico-chemical techniques such as pyrene fluorescence spectroscopy, differential scanning calorimetry, and ionic permeability were used to investigate the T(is)-lipid association. Results indicate that this system is an excellent one in which to study the boundary lipid phenomenon. In addition, T(is) association with lipid bilayers is being correlated with the natural state of glycophorin in the red cell membrane.

Interaction between glycophorin and phospholipids in recombined systems.

Both the MN-glycoprotein from human erythrocytes and the hydrophobic fragment from the protein isolated with trypsin treatment, T(is), have been recombined with egg phosphatidylcholine in bilayers at various phospholipid/protein ratios. In order to investigate the effect of the protein on the phospholipid headgroups, 31P nuclear magnetic resonance spectra were obtained with the MN-glycoprotein recombined with egg phosphatidylcholine, which revealed two classes of phospholipid environments, one immobilized and one not immobilized. Electron spin resonance (ESR) of fatty acid methyl ester spin labels provided supporting evidence. Computer analysis of the ESR spectra indicate that 4-5 moles of phospholipid are immobilized per mole of protein over a wide range of lipid-to-protein ratios. The immobilization of the phospholipids appears mediated by both the polar headgroups and the hydrocarbon tails of the phospholipid.

Kinetics and mechanism of dissociation of zinc ion from carbonic anhydrase.

The kinetics of dissociation of Zn2+ from the metalloenzyme carbonic anhydrase was measured over a range of pH, temperature, and acetate concentration. The rate of dissociation is extremely slow at neutral pH (t1/2 approximately 3) years, 4 degrees C), but increases in almost direct proportion to the hydrogen ion concentration and is enhanced in the presence of 1,10-phenanthroline or acetate. The thermodynamic stability of the zinc-apoenzyme complex was determined over a range of pH from rate data on binding and dissociation (stability constants 10(9)-10(11) M-1, 25 degrees C). The great stability of the complex and slow exchange of the apoenzyme ligand is attributed, at least in part, to the rigidity of the multidentate protein ligand.

Studies of synthetic peptide analogs of the amphipathic helix. Effect of charged amino acid residue topography on lipid affinity.

The amphipathic helix hypothesis for plasma lipoproteins was investigated using synthetic peptides. The lipid-associating properties of two potentially amphipathic model peptides and two analogs were studied by incubating synthetic peptides with small unilamellar vesicles and protein-lipid association examined by equilibrium density centrifugation, leakage of liposome-entrapped fluorescence compounds, intrinsic tryptophan fluorescence, and circular dichroism spectroscopy. The analog peptides were designed to determine the significance of the number and specific location of the charged residues in amphipathic domains of plasma lipoproteins to protein-lipid association. Based on the four procedures used to examine protein-lipid interactions, the two model peptides (18Aa, 18As) were found to associate strongly with liposomes; the two analog peptides (18As1, 18Asr), differing only with respect to the number and/or position of their charged residues, failed to demonstrate similar lipid binding properties. These findings support the earlier suggestions of the importance of the charged residues, but do not define the precise mechanisms involved. Such amino acids may help initiate the lipid-protein association by electrostatic interactions, contribute to the hydrophobicity of the nonpolar face of the helix by the acyl portion of lysine and arginine, and/or complement the charge distribution in the polar head regions of the phospholipid molecules.