Doctoral Dissertations

Date of Award

3-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Ernesto Freire

Abstract

The interaction of two soluble peptides, methionine enkephalin and the leader peptide of ornithine transcarbamylase, with phospholipid bilayers has been investigated using high sensitivity differential scanning calorimetry (DSC), fluorescence spectroscopy, electron microscopy, and enzymatic probes. In the case of methionine enkephalin, a preferential association with the ganglioside Gd1a was demonstrated. DSC studies indicated that methionine enkephalin concentrations as low as 10-5 M are adequate to induce phase separation in phospholipid vesicles containing Gd1a. Since there was no significant decrease in the enthalpy associated with the gel-to-1iquid crystalline phase transition of these vesicles, a headgroup interaction was predicted. Parameters calculated from the time-resolved decay curves of the fluorescence anisotropy of diphenylhexatriene (DPH) incorporated into phospholipid-ganglioside vesicles gave no indication of involvement of the hydrocarbon chains. The interaction of the neuropeptide and Gd1a was also demonstrated by enzymatic studies with tyrosinase and sialidase. The enkephalin is protected from tyrosinase oxidation by the ganglioside; conversely, methionine enkephalin will protect the ganglioside from sialidase hydrolysis. Sialidase studies performed with the soluble substrate sialyllactose provided further indication of ganglioside headgroup involvement. Both the tyrosinase and sialidase experiments gave Kd's for the methionine enkephalin-ganglioside Gd1a interaction in the range of 10-5 M, which is in agreement with the effect seen with the DSC.

The behavior of the leader peptide of the mitochondria] matrix enzyme, ornithine transcarbamylase, with phospholipid bilayers was also investigated. The temperature dependence of the steady-state flourescence anisotropy of DPH incorporated into lipid bilayers indicated that the peptide would cause a shift in the transition temperature (Tm) of negatively-charged vesicles, but not neutral vesicles; an increase in the anisotropy below the transition temperature of the negatively-charged vesicles was also noted. This shift in Tm was confirmed by DSC studies which showed that the peptide induced the formation of a second melting peak at the higher temperature. To investigate the origin of this second peak, negative stain electron microscopy of DPPC-brain PS and OPPC small unilamellar vesicles with the leader peptide was performed. From these, it was seen that peptide-lipid mole ratios as low as 1/200 were sufficient to cause fusion of negatively-charged vesicles in a temperature-dependent manner; the neutral OPPC vesicles did not fuse. Thus, the membrane physical state as well as the membrane charge is important in the consequences of the peptide-membrane association.

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