322

Polymyxin binding to charged p h o s p h o l i p i d bilayer membranes: A cooperative l i p i d - p r o t e i n - i n t e r a c t i o n F. Sixl and H.-J.

Galla

U n i v e r s i t ~ t Ulm, A b t e i l u n g f~r Biophysik, D-7900 Ulm (Donau), Germany

Oberer E s e l s b e r g

We have investigated the interaction between negatively charged p h o s p h o l i p i d bilayer membranes and the peptide antibiotic polymyxin-B. The m o l e c u l a r process of p o l y m y x i n binding was elucidated from the lipid phase transition curves. The o u t s t a n d i n g result for p h o s p h a t i d i c acid membranes was the d e t e r m i n a t i o n of a cooperative binding. The c o o p e r a t i v i t y of the binding was c o n t r o l l e d by pH and ion c o n c e n t r a t i o n of the buffer solution. The formation of a l i p i d - p e p t i d e - d o m a i n was established as a consequence of the antibiotic m o l e c u l a r structure (I). The strong binding of p o l y m y x i n to negatively charged lipid bilayer membranes is caused by both hydrophobic as well as e l e c t r o s t a t i c interaction. The packing density of the lipid membrane was found to control the b i n d i n g process thus showing a Langmuir- or cooperative type of binding curve. In case of a cooperative binding we established a modell for the domain structure. From calorimetric data we could estimate the interaction energy w i t h i n the polymyxin phosphatidic acid domain as well as the domain size. Competitive binding experiments with Ca 2+ were performed (2). Binding of p o l y m y x i n - B to mixed d i p a l m i t o y l p h o s p h a t i d i c acid/ d i s t e a r o y l - p h o s p h a t i d y l c h o l i n e membranes led to a phase separation. Domains of n o l y m y x i n - b o u n d p h o s p h a t i d i c acid were formed. Ca2+-ions were found to be a strong competitor in d i s p l a c i n g p o l y m y x i n from the complex in the mixed m e m b r a n e system. Comnlete d i s p l a c e m e n t was obtained at DH 9.0. With decreasing pH value Ca 2+ became a less strong competitor and was ineffective at pH 5.0. Binding of polymyxin was also observed to d i p a l m i t o y l p h o s p h a t i d y l c l y c e r o l membranes. I n c o r p o r a t i o n of polymyxin lowerd the lipid phase transition by 10 ~ C. One polymyxin was found to bind five p h o s p h a t i d y l g ! y c e r o l molecules. The binding curve was determined and in contrast to p h o s p h a t i d i c acid membranes, a n o n - c o o p e r a t i v e binding could be established. Addition of Ca 2+ decreased the amount of p h o s p h a t i d y l g l y c e r o l bound to p o l y m y x i n by about 20 %. No complete d i s p l a c e m e n t was achieved even at a ten-fold excess of Ca2+-ions with respect to phosphatidylglycerol. I) F. Sixl and H.-J. Galla, Biochim. 320-330 2) F. Sixl and H.-J. Galla, Biochim. 626-635

Biophys.

Acta 557

(1979)

Biophys.

Acta 643

(1981)