Characterization of the adhesion plaque proteins vinculin and talin: Their localization and interaction with each other and with actin in vitro

Thesis/Dissertation · Fri Jan 01 04:00:00 EST 1988

OSTI ID:5160699

Groesch, M E

We have studied vinculin and talin, adhesion plaque proteins that have been proposed to be involved in linking the cytoskeleton to the membrane. Polyclonal antibodies were raised against vinculin and talin and used in immunofluorescence studies to confirm that talin and vinculin are localized in adhesion plaques. We used falling ball viscometry to try to detect interactions between polymerized actin and vinculin and/or talin in vitro and found no evidence of any interaction, suggesting that neither protein directly links actin to the membrane. Since it was known that vinculin and talin interact, studies were carried out to localize the talin-binding site on the vinculin molecule. Vinculin was digested with Staphylococcus aureus V8 protease into two major fragments of 85 kDa and 30 kDa. We were able to purify both of the V8 protease-generated vinculin fragments. Nitrocellulose overlays with {sup 125}I-talin and {sup 125}I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of {sup 125}I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin.

• Research Organization: Purdue Univ., Lafayette, IN (USA)
• OSTI ID: 5160699
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACTIN
ADHESION
ANTIBODIES
BACTERIA
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL LOCALIZATION
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
FLUORESCENCE
HYDROLASES
IN VITRO
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LUMINESCENCE
MEMBRANE PROTEINS
MEMBRANES
MICROORGANISMS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
STAPHYLOCOCCUS
TRACER TECHNIQUES