Stepwise changes in crossbridge state and sarcomere length: do lattice constraints play a critical role?

Observed abrupt and stepwise changes in crossbridge state induced by MgPPi in skeletal fibers using spin label techniques as well as stepwise changes in sarcomere length in actively contracting fibers are considered in terms of a model based on the principles of catastrophe theory and nucleation of structural transitions in mechanically constrained polymeric assemblies.

Tryptic proteolysis of coupling factor-latent ATPase from mycobacterium phlei. Theoretical modeling of structure-function relationships.

Trypsin treatment of solubilized coupling factor-latent ATPase from Mycobacterium phlei alters its subunit structure and functional properties. This coupling factor exhibits ATPase activity following trypsin treatment. Concurrently, both the ability of the enzyme to rebind to membranes depleted of coupling factor and its capacity for coupled phosphorylation are lost. The native alpha (64 000 dalton) subunit undergoes limited proteolytic digestion, and the delta (14 000 dalton) subunit is partially lost. During the course of tryptic proteolysis, the coupling factor molecule may exist in one of ten unique structural state (e.g. the native, ATPase-inactive molecule exists in the alpha alpha alpha state). Rigorous analysis of the experimental data by theoretical modeling provided information concerning the intermediate structural states leading to the fully ATPase-activated alpha" alpha" alpha" state under different conditions of trypsin treatment. The theoretical models of structure-function relationships that best-represented the experimental data predicted that the native coupling factor molecule contains three copies of the alpha (64 000 dalton) form of the alpha subunit, that the alpha" (58 000 dalton) alpha subunit species contributes maximally and the alpha' (61 000 dalton) form about half-maximally to ATPase activity, that membrane rebinding ability is proportional to the number of native alpha subunits in the enzyme, and that at least one native alpha subunit/molecule is required for full expression of coupled phosphorylation. These results indicate an essential role for the alpha subunit in the regulation of ATPase activity and in the ability of the solubilized coupling factor to rebind to depleted membranes.

Limited proteolysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents.

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solubilized coupling factor with chymotrypsin rapidly produced an A'-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the APTase enzyme. Secondary chymotryptic cleavage yielded an A"-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at -20degreeC in the presence of 4 mM MgCl2 with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4 degrees C in the presence or absence of MgCl2 both decreased ATPase activity and generated A'-type alpha subunit species. Since presence was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparantly located within the same small segment of alpha subunit polypeptide chain.