alpha-chymotrypsin and 3-(trifluoromethyl)-3-(3-iodophenyl)diazirine

CTP:phosphocholine cytidylyltransferase (CT), the rate controlling enzyme in phosphatidylcholine biosynthesis, is activated by reversible membrane binding. To investigate the membrane binding mechanism of CT, we have used the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[l25I]iodophenyl)diazirine ([125I]TID). Association of CT with phosphatidylcholine/oleic acid (1:1) vesicles was first demonstrated by gel filtration analysis. Upon irradiation, CT was covalently labeled by [125I]TID presented in phosphatidylcholine/oleic acid vesicles. This demonstrates an intercalation of part of the protein into the hydrophobic core of the membrane. To identify the membrane-embedded domain, the chymotrypsin digestion products of [125I]TID labeled CT were analysed. Chymotrypsin digestion produced a set of previously defined N-terminal fragments (Craig, L., Johnson, J.E. and Cornell, R.B. (1994) J. Biol. Chem. 269, 3311), as well as several small C-terminal fragments which react with an anti-peptide antibody raised against the proposed amphipathic alpha-helix. All fragments containing the amphipathic helical region of the enzyme had [125I]TID label associated, while the chymotryptic fragment which lacked this region was not highly labeled. Similar fragment labeling patterns were produced when [125I]TID was presented in phosphatidylcholine/oleic acid or phosphatidylcholine/diacylglycerol vesicles, suggesting that the same domain of CT mediates binding to membranes containing either of the two lipid activators. A 62-residue synthetic peptide corresponding in sequence to the amphipathic helical region of CT was labeled with [125I]TID, demonstrating its ability to intercalate independently of the rest of the protein. These results indicate a membrane-binding mechanism for cytidylyltransferase involving the intercalation of the amphipathic alpha-helix region into the hydrophobic acyl chain core of the activating membrane.

Topics: Animals; Azirines; Cell Membrane; Choline-Phosphate Cytidylyltransferase; Chymotrypsin; Cross-Linking Reagents; Diglycerides; Membrane Lipids; Molecular Weight; Nucleotidyltransferases; Oleic Acid; Peptide Fragments; Peptides; Phosphatidylcholines; Photochemistry; Protein Binding; Protein Structure, Secondary; Rats; Recombinant Proteins; Sequence Analysis

Mitochondrially bound rat brain hexokinase was labeled with the photoactivatable reagent, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. This highly hydrophobic reagent is strongly partitioned into the hydrophobic environment of the membrane core, and thus selectively labels segments of a protein that penetrate this region of the membrane. Labeling of hexokinase was shown to be restricted to the N-terminal region of the molecule. Approximately 80% of the radiolabel was removed by treatment of the enzyme with chymotrypsin, which preferentially cleaves a hydrophobic 9-residue sequence at the extreme N-terminus of the enzyme, and it is considered likely that the remaining 20% was associated with two additional hydrophobic residues, immediately adjacent to this segment but not susceptible to cleavage by chymotrypsin. Labeling of the enzyme was shown to be dependent on maintenance of the association with the membrane. These results are consistent with a model in which binding of hexokinase involves insertion of an 11-residue hydrophobic N-terminal "tail," possibly existing in alpha-helical secondary structure, into the hydrophobic core of the membrane.

Topics: Affinity Labels; Animals; Azirines; Binding Sites; Brain; Chymotrypsin; Hexokinase; Lipid Bilayers; Membrane Proteins; Mitochondria; Porins; Rats; Solubility; Trypsin; Voltage-Dependent Anion Channels

The photoactivatable carbene precursor 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ( [125I]TID) was tested as a probe for labeling lipid-embedded segments of the proteins of pure membrane bound (Na+ + K+)-ATPase. The probe labeled the alpha-subunit (100 kDa), its major tryptic and chymotryptic fragments of 78 kDa, 58 kDa, and 46 kDa, and the beta-subunit (38 kDa) from within the lipid bilayer to nearly the same specific activity. The labeling was resistant to extensive proteolysis and the distribution of label among the proteolytic fragments and the two subunits was independent of a 47-fold variation in concentration of [125I]TID. The data show that several transmembrane segments are distributed along the sequence of the alpha-subunit and that also the beta-subunit traverses the bilayer. [125I]TID provides a more uniform labeling of the transmembrane segments of the alpha-subunit and beta-subunit than that obtained with other hydrophobic reagents. This will facilitate further studies of the primary structure and folding pattern of the Na+,K+-pump proteins in the membrane.

Topics: Animals; Azirines; Cell Membrane; Chymotrypsin; Iodine Radioisotopes; Kidney Medulla; Macromolecular Substances; Molecular Weight; Peptide Fragments; Protein Binding; Sodium-Potassium-Exchanging ATPase; Swine; Trypsin

The interaction of the opioid enkephalins and endorphins with lipid bilayers is largely unknown. Such interactions might, however, be important for understanding the molecular mechanisms of biological action. We have therefore studied the interaction of several enkephalins and of dynorphin-(1-13)-tridecapeptide (dynorphin1-13) with model membrane systems, using the extremely hydrophobic photolabel of J. Brunner and G. Semenza, (Biochemistry 20, 7174-7182 (1981)) 3-trifluoromethyl-3-(m[125I]iodophenyl)diazirine. By observing several limitations of the method, it was possible to characterize hydrophobic interactions of opioid peptides with liposomes prepared from egg yolk phosphatidylcholine (PC) plus dipalmitoylphosphatidic acid (PA), from brain phosphatidylserine (PS) alone, and from brain cerebroside sulfate (CS) alone, Dynorphine1-13 exhibited strong hydrophobic interactions through its N-terminal "message" segment which were potentiated by the "address" that itself remained in the aqueous phase. This behavior was consistent with the reported pharmacological potentiation. The enkephalins were generally weakly labeled in the PC/PA and PS systems. However, in the CS systems the preferentially mu agonists were labeled very strongly whereas the preferentially delta agonists were labeled more weakly yet. The kappa agonist, dynorphin1-13, was strongly, but more equally labeled in the three systems. Thus, there was a head group specificity that, in our series of compounds, correlated with opiate receptor sub-type specificity. The results were consistent with the behavior of the mu agonist, morphine.

Topics: Amino Acid Sequence; Azirines; Chymotrypsin; Dynorphins; Endorphins; Enkephalins; Lipid Bilayers; Peptide Fragments; Structure-Activity Relationship