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Biomedical subjects

C M Deber

Publications and source records attributed to C M Deber.

At least 73 records · Page 4Linked to original sources
PubMed1986

Calcium transport by ionophorous peptides in dog and human lymphocytes detected by quin-2 fluorescence.

Synthetic peptides of structure cyclo(Glu(OBz)-Sar-Gly-(N-R)Gly)2 (I), electrogenic Ca2+-selective carriers in phospholipid vesicle membranes, are shown to mediate the uptake of Ca2+ ions into the cytoplasm of dog and human lymphocytes. Ca2+ transport by DECYL-2E (I, R = n-decyl) - monitored by measurements of the fluorescence of an intracellular dye, quin-2 - occurred at a rate comparable to that produced by electroneutral Ca2+ ionophores ionomycin and Br-A23187. Fluorescence quenching experiments using Mn2+ suggested a greater selectivity by DECYL-2E for Ca2+/Mn2+ vs. the other two ionophores. The result that Ca2+ ions can traverse biological membranes bound in a neutral cavity consisting exclusively of peptide carbonyl ligands may imply the functional significance of binding sites of similar structures in membrane transport proteins.

Aminoquinolines↗
PubMed1986

Binding of human normal and multiple sclerosis-derived myelin basic protein to phospholipid vesicles: effects on membrane head group and bilayer regions.

The detailed interaction of human myelin basic protein (MBP) with charged lipids may be critical in organizing the myelin sheath into its biologically functional structure. Carbon-13 and phosphorus-31 nuclear magnetic resonance spectroscopy has been used to study this interaction by examining spectral consequences of additions of MBP to membrane preparations of the negatively charged lipid phosphatidylglycerol (PG). Lipid head group 13C and 31P linewidths were found to narrow upon addition of protein, while concomitant broadening was noted for bilayer carbon resonances. At intermediate MBP/PG ratios, two components in slow exchange on the NMR time scale (bulk PG and a protein-induced PG domain) were observed for the 13C resonance of the head group carbon atom adjacent to phosphate. These results, and other spectral evidence, suggested that head groups in free PG vesicles are motionally restricted by intermolecular interactions which are disrupted by competition with MBP Lys and Arg positively charged side chains. Titration of PG with the homopolypeptide poly-L-lysine produced comparable effects on PG 13C head group spectra, indicating that electrostatic attractions constitute the primary basis of the observed interactions. Vicinal and/or geminal 13C-31P coupling constants measured from the spectra of PG head group carbons were found to be essentially invariant for free PG in dimethyl sulfoxide solution, free PG vesicles, PG vesicles + MBP, and PG vesicles + poly-L-lysine. Comparison of the spectral effects induced in PG head group resonances by normal vs multiple sclerosis-derived MBP (MS-MBP) indicated that the MS-MBP is relatively less effective in converting PG to the protein-induced domain, a result which was attributed to increased protein self-aggregation arising from the reduced net positive character of the MS protein samples.

Chemical Phenomena↗
PubMed1986

Hypothesis about the function of membrane-buried proline residues in transport proteins.

In a survey of the bilayer-spanning regions of integral membrane proteins, membrane-buried proline residues were found in nearly all transport proteins examined, whereas membrane-buried regions of nontransport proteins were largely devoid of intramembranous proline residues. When amino acids from the complete sequences of representative sets of transport and nontransport membrane proteins were analyzed for the distribution of proline residues between aqueous vs. membranous domains, proline was shown to be selectively excluded from membranous domains of the nontransport proteins, in accord with expectation from energetic and structural considerations. In contrast, proline residues in transport proteins were evenly distributed between aqueous and membranous domains, consistent with the notion that functional membrane-buried proline residues are selectively included in transport proteins. As cis peptide bonds involving proline arise in proteins and have been implicated in protein dynamic processes, the cis-trans isomerization of an Xaa-Pro peptide bond (Xaa = unspecified amino acid) buried within the membrane--and the resulting redirection of the protein chain--is proposed to provide the reversible conformational change requisite for the regulation (opening/closing) of a transport channel. Parallel to this function, the relatively negative character of the carbonyl groups of Xaa-Pro peptide bonds may promote their participation as intramembranous liganding sites for positive species in proton/cation transport processes.

Amino Acid Sequence↗
PubMed1985

Structure and function of the proline-rich region of myelin basic protein.

Myelin basic protein (MBP)--the major extrinsic membrane protein of central nervous system myelin--from several species contains a rarely encountered highly conserved triproline segment as residues 99-101 of its 170-residue sequence. Cis peptide bonds are known to arise at X-Pro junctions in proteins and may be of functional significance in protein folding, chain reversal, and/or maintenance of tertiary structure. We have examined the conformation of this proline-rich region using principally 13C nuclear magnetic resonance spectroscopy (125 MHz) both in intact bovine MBP and in several MBP fragment peptides which we synthesized, including octapeptide 97-104 (Arg-Thr-Pro-Pro-Pro-Ser-Gln-Gly). Results suggested an all-trans conformation in aqueous solution for the triproline segment in MBP hexapeptide (99-104), heptapeptide (98-104), and octapeptide. Comparison with the 13C spectrum of intact MBP (125 MHz) suggested that the proline-rich region, as well as all other X-Pro MBP peptide junctures, was also essentially all trans in aqueous solution. Although experiments in which octapeptide 97-104 was bound to a lipid preparation (4:1 dipalmitoylphosphatidylcholine/dimyristoylphosphatidic acid) demonstrated that cis-proline bonds do arise (to the extent of ca. 5%) in the membrane environment, a role of linear chain propagation is suggested for the triproline segment of myelin basic protein.

Amino Acid Sequence↗
PubMed1985

Bromo-A23187: a nonfluorescent calcium ionophore for use with fluorescent probes.

4-Bromo-A23187, a halogenated analog of the widely studied divalent cation ionophore A23187, is a nonfluorescent Ca2+ ionophore suitable for use in the calibration of cytoplasmic free Ca2+ by fluorescent probes. Br-A23187 is shown to saturate Ca2+ sites in quin-2-loaded rat thymic lymphocytes in a manner essentially identical to ionomycin.

Aminoquinolines↗
PubMed1984

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment.

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.

Dimethyl Sulfoxide↗
PubMed1984

NMR investigation of the charge isomers of bovine myelin basic protein.

Myelin basic protein (MBP) isolated from bovine white matter is obtained as a mixture of molecules which can be separated by cation-exchange chromatography at basic pH into three or more charge isomers. The three principal charge isomers of the microheterogeneous myelin basic protein have been isolated, and compared individually by high-resolution H NMR spectroscopy (360 and 400 MHz). In addition to confirming sources of MBP charge microheterogeneity such as fractional deamidation of Gln and loss of C-terminal Arg, NMR difference and spin-echo spectra further suggested (i) the presence of significant oxidation of (both) MBP Met residues to methionine sulfoxide; (ii) the three charge isomers contain equal ratios and absolute contents of mono- and dimethylated Arg; and (iii) the most-cationic isomer is deficient in its content of a putative extra Ala residue vs the other two isomers. Spectral analysis suggested that each MBP charge isomer is itself not a unique molecule, but more likely a mixture of molecules of equal net charge which are modified at any of the indicated functional side chains throughout the 169-residue protein. The results are discussed with respect to the possible consequences of MBP microheterogeneity to protein conformation and function.

Animals↗
PubMed1984

Surface accessibility of 13C-labeled lysine residues in membrane-bound myelin basic protein.

Surface-exposed regions of membrane-bound myelin basic protein--the major extrinsic membrane protein of central nervous system myelin--have been implicated as possible antigenic sites in diseased myelin. With the goal of determining the extent and nature of these regions, we have prepared basic protein modified with 13CH3-enriched acetyl groups at 7 of its 13 lysine residues. The resulting protein was placed in a membrane environment and studied by NMR spectroscopy to determine the location and rates of molecular motion of the labeled side chains with respect to lipid bilayers of the membrane. When 13C NMR spectra were obtained of the acetylated protein bound to multilamellar vesicles prepared from dimyristoylphosphatidic acid in the gel state (T = 33 degrees C), conditions under which reduced motion in the lipid bilayer broadens methylene and methyl 13C resonances of the membrane beyond detection (i.e. greater than 75-100 Hz), line widths of membrane-bound protein were measured to be 7.8 Hz, an increase of 4 Hz versus free protein. A reduction of 25-30% in integrated intensity observed in protein acetyl resonances upon membrane interaction was shown to be attributable to a population of protein-aggregated liposomes whose resonances were similarly too broad to be observed. Thus, the epsilon-acetyllysyl probes distributed throughout the protein do not penetrate the dimyristoylphosphatidic acid bilayer, but must reside in the interstitial aqueous spaces at or between membrane surfaces. These findings suggest an overall surface accessibility of membrane-bound myelin basic protein and are therefore incompatible with a model for the protein involving membrane-embedded loops or regions of functional significance.

Acetic Anhydrides↗
PubMed1984

Role of membrane lipids in peptide hormone function: binding of enkephalins to micelles.

In the course of their biological function, peptide hormones must be transferred from an aqueous phase to the lipid-rich environment of their membrane-bound receptor proteins. We have investigated the possible influence of phospholipids in this process, using 360-MHz 1H and 90-MHz 13C NMR spectroscopy to examine the association of the opioid peptides [Met]- and [Leu]enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) with phospholipid micelles. Binding of peptides to lipid was monitored in NMR spectra by selective chemical shift movements (e.g., the Phe aromatic ring protons) and residue-specific line broadening (e.g., of Met/Leu carbonyl- and alpha-carbon resonances). Results established that the zwitterionic hormones associate hydrophobically both with a neutral lipid (lysophosphatidylcholine) and (also electrostatically) with a negative lipid (lysophosphatidylglycerol). An association constant of Ka = 3.7 X 10(1) M-1 was calculated for the hydrophobic binding of enkephalin to lysophosphatidylcholine. NMR data suggested that enkephalin binds to the lipid with Met/Leu, Phe, and likely Tyr side-chain substituents associated with nonpolar interior regions of the micelle, whereas the COOH-terminal carboxylate moiety of the peptide is located in the surface of the lipid particle. An "attraction-interaction" model is proposed for hormone-lipid association wherein negative lipids attract the hormone electrostatically, while site-specific hydrophobic contacts facilitate its entry, concentration, and orientation into the lipid phase.

Enkephalin, Leucine↗
PubMed1982

Cation transport properties of a synthetic Ca2+-selective peptide ionophore in phospholipid and sarcoplasmic reticulum vesicles.

Transport by the synthetic cyclic peptide ionophore CYCLEX-2E (Deber, C.M. Young, M.E.M., and Tom-Kun, J. (1980) Biochemistry 19, 6194-6198), which in contrast to Ca2+ ionophore A23187 contains no ionizable protons, has been studied with respect to Ca2+ and Na+ transport, and the involvement of exchanged, or counter-transported ions during the transport process. CYCLEX-2E was found to equilibrate Na+ and Ca2+ gradients across phospholipid vesicle membranes. Experiments using the indicator dye Arsenazo III established that calcium ions were indeed reaching the aqueous intravesicular compartments. Absence of metal cations in the external buffer slowed, but did not eliminate, the efflux of Ca2+ from phosphatidylcholine vesicles. As an example of its activity in a biological membrane, CYCLEX-2E was shown to be capable of producing Ca2+ efflux from sarcoplasmic reticulum vesicles which has been loaded with Ca2+ in an ATP-dependent manner. The overall results suggest that in transport by synthetic peptide ionophores typified by CYCLEX-2E, electroneutrality is achieved either through (a) peptide-mediated compensating (but not coupled) fluxes of other cations, or where this is not an option, by (b) transmembrane diffusion of permeant ions such as H+, OH-, or Cl-.

Animals↗
PubMed1982

Binding of myelin basic protein to phospholipid micelles.

Resonances of 1H NMR spectra (360 MHz) due to several individual substituents of myelin basic protein reconstituted into phospholipid micelles have been resolved and assigned. With the aid of spin echo techniques to remove overlapping signals in the region of 2-3 ppm, singlet resonances due to the two myelin basic protein methionine S--CH3 groups (Met-20 and Met-167) and the Ala-1 N-acetyl methyl group were observed. In the presence of sufficient lysophosphatidylcholine to fully bind the protein, differential broadening occurred for Met S--CH3 resonances; Met-20 lost about 40% of its original integrated intensity (while displaying no significant line broadening) but Met-167 and N-acetyl-Ala-1 resonances did not appear to be influenced by lipid. The differential broadening of the Met-20 (versus the Met-167 and N-acetyl-Ala-1) resonances was proposed to arise from (a) specific restriction to local molecular motion of the Met-20 (but not Met-167) side chain (i.e. due to its penetration into the hydrophobic micelle lipid matrix or its proximity to specific protein/lipid electrostatic sites); or (b) transmitted dynamic effects of overall protein binding to micellar particles (due to the relatively fewer degrees of rotational freedom available to Met-20 versus N-acetyl-Ala-1 and Met-167 near protein termini). Evidence favoring the latter alternative is presented.

Animals↗
PubMed1980

Synthetic cation transport peptides: calcium transport across phospholipid membranes.

Molecular aspects of peptide-mediated calcium transport are examined through the study of the cation transport properties of a series of synthetic cyclic octapeptides. These peptides, of general structure cyclo[Glu(OR1)-Sar-Gly-(N-R2)Gly]2 (R1 = H or benzyl ester; R2 = cyclohexyl, n-hexyl, or n-decyl) (and an Asp analogue), contain central binding cavities of geometry and dimensions similar to calcium-binding sites in proteins. Transport in Pressman cells ("thick liquid membranes") demonstrated the ionophorous activity of the synthetic peptides; among physiologically abundant cations, the order of selectivity was Ca2+ greater than Na+, K+ much greater than Mg2+. Cation competition studies further showed that cyclo[Glu(OBz)-Sar-Gly-(N-cyclohexyl)Gly]2 (CYCLEX-2E) is essentially a calcium-specific transport peptide whenever calcium is present. When the CYCLEX-2E peptide was added to a suspension of 45Ca2+-loaded sonicated phosphatidylcholine (PC) vesicles in a dialysis sac, the vesicles were completely emptied of internal calcium. Controls using [14C]sucrose established that CYCLEX-2E caused no nonspecific membrane damage. Calcium efflux experiments using several salts of calcium (including 36C1-, [14C]acetate, [14C]succinate, and 35SO4(2-)) suggested that these anions do not specifically accompany the Ca2+-peptide active transporting species across the phospholipid membrane. However, when 45Ca2+-loaded PC vesicles were suspended in mental-free buffer and treated with CYCLEX-2E peptide, calcium efflux did not occur until calcium or sodium chloride was added to the external medium.

Binding Sites↗