Date of Award

1-1-1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

Clathrin coated vesicles (CCVs) are complex organelles reported to bind specific glycolytic enzymes. The purpose of this study was to further biochemically characterize the interactions of selected enzymes with CCV's by conventional and new techniques.Purified brain CCVs were observed to retain, in varying degrees, endogenous glycolytic enzymes, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) having the highest specific activities. Furthermore, Scatchard and Hill plot analyses of saturation binding data suggested that the binding of pure aldolase (ALD) and GAPDH involved heterogenous binding sites or cooperativity. Moreover, increasing concentrations of added ALD could not completely release bound GAPDH from CCVs, suggesting that these enzymes share a common binding site but may have other unique sites. Approximately, 72% of the total ALD activity and 63% of the total GAPDH activity was bound under physiological salt concentrations if an excluded volume effector, polyethylene glycol, was present in the assay.Ligand blotting was used to further investigate the binding of GAPDH to CCVs with the intention of identifying potential GAPDH binding polypeptides. Immobilized CCV polypeptides were probed with GAPDH, the ligand, followed by immunoblotting with a polyclonal anti-GAPDH. Four intense bands were observed on the blot and were identified as the clathrin heavy chain, a polypeptide with a molecular weight of 100 kDa, and alpha and beta tubulin.Lastly, native agarose gel electrophoresis (AGE) was thoroughly examined as a potential technique to qualitatively identify complexes between glycolytic enzymes and CCVs (or their subcomponents), tubulin, and actin. The technique was based on the differential electrophoretic mobilities observed between complexes and free enzyme. Using AGE, GAPDH was observed to saturate its binding sites on tubulin. Aldolase, PK, and lactate dehydrogenase (LDH) bound tubulin, and the interactions are suggested to be specific. Both stripped vesicles and a coat protein fraction bound ALD, GAPDH, PK, and LDH suggesting that these CCV subcomponents contain binding sites for each enzyme. Additionally, a protease-sensitive region on the stripped vesicles was identified to bind ALD and PK.

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