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.

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.

The location of secondary structure in histone H4.

A total of eight peptides cleaved from calf thymus histone H4 have been studied at several ionic strengths by circular dichroic, infrared and nuclear magnetic resonance spectroscopies to follow the formation of alpha helix, beta structure and self-aggregates. The results are compared with data obtained previously on three other peptides and on the intact molecule in order to define the location of secondary structure in histone H4. It is concluded that there are two alpha-helical sections, the first from residues 55 to 67 and the second of about 12 residues in the region between residues 70 to 90. beta-Structure formation takes place only in the C-terminal part of the intact H4 molecule. Nuclear magnetic resonance studies of the peptides prove that it is the basic N-terminal regions of histone H4 that remain free when the molecule self-aggregates.