STUDIES OF THE TROPONIN REGULATION OF MUSCLE CONTRACTION

  • Potter, James D., (PI)

Project: Research project

Description

The regulation of cardiac and skeletal muscle contraction by Ca++ involves
a series of complex protein-protein interactions between the three subunits
(TnT, the tropomyosin (Tm) binding subunit, TnI, the inhibitory subunit,
and TnC, the Ca++ binding subunit) of troponin (Tn), the two subunits of
tropamyosin and actin. Quite a bit is known about the subunit interactions
between the isolated Tn component pairs, and this information has been
extrapolated back to the intact complex, although there is no easy way to
test the validity of these extrapolations. One way we propose to study
these interactions and their role in muscle regulation is to use monoclonal
antibodies as specific probes of these protein-protein interactions. The
basic strategy in this project will be to initially prepare monoclonal
antibodies against the three individual Tn subunits (and selected fragments
of the subunits) and then characterize them with regards to their
specificity, stoichiometry, and affinity. We will also determine as nearly
as possible the location of the various antigenic determinants. Since the
amino acid sequence of all of the Tn subunits is known and a variety of
proteolytic fragments are available from them, it should be possible to
narrow down the regions of the proteins which make up the antigenic
determinant using a variety of techniques. We will also determine whether
or not these different antibodies can bind to these antigenic sites on the
individual proteins in the various reconstituted systems (e.g. TnC-TnI,
TnI-TnT, TnT-TnC, Tn, Tn.Tm, Tn.Tm.Actin, etc.) in different conformational
states (e.g., Ca++, extent of filament overlap, rigor, etc.) We will also
test to see if any of these antibodies affect the function of Tn by testing
them against myofibrils (ATPase assays) and skinned fibers (tension
development). In this way it should be possible to map regions of the
various Tn subunits that are functionally important in addition to those
regions which are/are not available for antibody binding in the different
protein complexes and/or conformational states. Other groups (1, 2) have
attempted to ask similar questions about Tn subunit interactions through
the use of either protein fragments or chemical reactivity studies, methods
which probably substantially alter the native structure of the proteins
and, in addition, only give information about the interactions and not
about function. In contrast, the approach designed in this application
should yield information about Tn subunit interactions in the native state
as well as their function.
StatusFinished
Effective start/end date7/1/836/30/87

Funding

  • National Institutes of Health

Fingerprint

Troponin
Muscle
Tropomyosin
Proteins
Antibodies
Actins
Chemical reactivity
Extrapolation
Stoichiometry
Adenosine Triphosphatases
Epitopes
Assays

ASJC

  • Medicine(all)