Date of Award:

5-1-1980

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Life Sciences:Biology

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Committee

G. Miller

Committee

F. Post

Committee

T. Farley

Committee

S. Oberg

Abstract

Radioisotopic techniques were used to identify, quantitate and elucidate the topology of phospholipids in the photosynthetic membrane of Rhodopseudorronas sphaeroides. Two major phospholipids, phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), were identified in the lipids extracted from chromatophores, spheroplast-derived vesicles (SDV's) and whole cells. PE and PG were found to comprise 63 percent and 35 percent of total phospholipids respectively. A third phospholipid comprising about two percent of the total phospholipids was not identified. The topological distribution of phospholipids in the photosynthetic membrane was investigated using phospholipase hydrolysis and trinitrobenzene sulfonic acid (TNBS) labelling methods on chromatophores and SDV's. Under non-lytic conditions, treatment of chromatophores with phospholipase A2 resulted in hydrolysis of 84 percent of the total PE and 73 percent of the total PG. Treatment of the SDV' s under the same conditions resulted in hydrolysis of 66 percent of the total PE and 14 percent of the total PG. Thus, the hydrolysis of PG in chromatophores and SDV's was complementary (73 percent and 14 percent of total PG was located at the inner and outer surfaces of the cytoplasmic membrane, respectively). On the other hand, the hydrolysis of PE in both membrane systems was not complementary. These results were interpreted as due most likely to the transmembrane movement of PE from the inner to the outer surface of the membrane vesicles. This conclusion was supported by the observation that 9 percent of the total PE originally located at the inner surface of chromatophores became accessible to phospholipase hydrolysis on the outer surface. TNBS was used to elucidate the topology of PE in chromatophores and SDV's. A kinetic analysis of the reaction of TNBS with the PE of chromatophores and SDV's was performed. The percent of unreacted PE versus the time of reaction was plotted in a semilogarithmic scale. The semilogarithmic plot was biphasic, revealing fast- and slow-reacting PE components. The fast-reacting component represents the fraction of PE located at the outer surface of the vesicles, while the slow-reacting component represents the fraction of PE located at the inner surface or embedded in the membrane. Analyzed in this way, it was found that 35 percent of the total PE was located at the outer surface of the chromatophores, 61 percent was located at the inner surface and 6 percent was not labelled. For SDV's, 55 percent of total PE was located at the outer surface, 32 percent was located at the inner surface and 13 percent was not labelled. These results were interpreted as follows: 55 percent of the total PE is located at the outer surface and 35 percent is located at the inner surface of the cytoplasmic membrane of R. sphaeroides.

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