The concentrations of glucose 1,6-bisphosphate and other regulatory metabolites, and the activities of enzymes of the glycogen metabolism in the perfused rabbit psoas muscle

The following parameters were determined in the rabbit psoas muscle after perfusion in the presence of either insulin, propranolol, or isoproterenol: Concentrations of cyclic AMP, glucose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose-1-phosphate, glucose 6-phosphate, and fructose-1,6-bisphosphate. Maximum and "regulatory" activities of the enzymes glycogen phosphorylase, glycogen synthase, phosphofructokinase, and histone-phosphorylating protein kinase.     

Structural characterization of the binding of Myosin*ADP*Pi to actin in permeabilized rabbit psoas muscle

When myosin is attached to actin in a muscle cell, various structures in the filaments are formed. The two strongly bound states (A*M*ADP and A*M) and the weakly bound A*M*ATP states are reasonably well understood. The orientation of the strongly bound myosin heads is uniform ("stereospecific" attachment), and the attached heads exhibit little spatial fluctuation. In the prehydrolysis weakly bound A*M*ATP state, the orientations of the attached myosin heads assume a wide range of azimuthal and axial angles, indicating considerable flexibility in the myosin head. The structure of the other weakly bound state, A*M*ADP*P(i), however, is poorly understood. This state is thought to be the critical pre-power-stroke state, poised to make the transition to the strongly binding, force-generating states, and hence it is of particular interest for understanding the mechanism of contraction. However, because of the low affinity between myosin and actin in the A*M*ADP*P(i) state, the structure of this state has eluded determination both in isolated form and in muscle cells. With the knowledge recently gained in the structures of the weakly binding M*ATP, M*ADP*P(i) states and the weakly attached A*M*ATP state in muscle fibers, it is now feasible to delineate the in vivo structure of the attached state of A*M*ADP*P(i). The series of experiments presented in this article were carried out under relaxing conditions at 25 degrees C, where approximately 95% of the myosin heads in the skinned rabbit psoas muscle contain the hydrolysis products. The affinity for actin is enhanced by adding polyethylene glycol (PEG) or by lowering the ionic strength in the bathing solution. Solution kinetics and binding constants were determined in the presence and in the absence of PEG. When the binding between actin and myosin was increased, both the myosin layer lines and the actin layer lines increased in intensity, but the intensity profiles did not change. The configuration (mode) of attachment in the A*M*ADP*P(i) state is thus unique among the intermediate attached states of the cross-bridge ATP hydrolysis cycle. One of the simplest explanations is that both myosin filaments and actin filaments are stabilized (e.g., undergo reduced spatial fluctuations) by the attachment. The alignment of the myosin heads in the thick filaments and the alignment of the actin monomers in the thin filaments are improved as a result. The compact atomic structure of M*ADP*P(i) with strongly coupled domains may contribute to the unique attachment configuration: the "primed" myosin heads may function as "transient struts" when attached to the thin filaments.     

Stimulation of autophosphorylation of rabbit skeletal muscle phosphorylase kinase by glycogen synthase on glycogen particles

Glycogen synthase stimulated the autophosphorylation and autoactivation of phosphorylase kinase from rabbit skeletal muscle. This stimulation was additive to that by glycogen and the reaction was dependent on Ca2+. The effect by glycogen synthase was maximum within the activity ratio (the activity of enzyme without glucose-6-P divided by the activity with 10 mM glucose-6-P) of 0.3 and over 0.3 it was rather inhibitory. The results suggest that autophosphorylation of phosphorylase kinase in the presence of glycogen synthase on glycogen particles may be an important regulatory mechanism of glycogen metabolism in skeletal muscle.     

Compartmentalized protein phosphorylation/dephosphorylation in glycogen particles from rabbit skeletal muscle

Activation of phosphorylase in intact glycogen particles from skeletal muscle by Ca2+ and MgATP is known as flash activation. By using [gamma-32P]ATP to monitor protein phosphorylation, we have demonstrated that there is, coincident with phosphorylase activation and inactivation, coordinated phosphorylation/dephosphorylation of phosphorylase, glycogen synthase, the beta-subunit of phosphorylase kinase and proteins of Mr = 43,000 and 32,000. Our results show that within the glycogen particle phosphorylase kinase and type-1 protein phosphatase are organized to allow access to a set of protein components. This arrangement may contribute to the reciprocal regulation of their activities.     

Changes associated with glycolytic-enzyme binding in the equatorial X-ray-diffraction pattern of glycerinated rabbit psoas muscle.

The binding of fructose biphosphate aldolase to the thin filaments of glycerinated rabbit psoas muscle produces a significant change in its low-angle X-ray-diffraction pattern. The intensity of the (11) reflection relative to that of the (10) reflection increases by 26 +/- 3% (mean +/- S.E.M.), which is consistent with the increase in the mass of the thin filaments produced by enzyme binding. A similar effect is found with a mixture of aldolase and glyceraldehyde 3-phosphate dehydrogenase. The significance of the change in intensity is considered with reference to the interpretation of the equatorial patterns obtained from muscles in different physiological states. The magnitude of the increase in the relative intensity of the (11) reflection is lower than that observed between relaxed and contracting muscle and does not bring into question the interpretation linking changes in these patterns to cross-bridge movement. However, the effect due to enzyme binding may be important when making detailed interpretations of these changes. It may also be related to an unusual pattern sometimes observed in cardiac muscle.     

Heparin-activated protein kinase from rabbit muscle: relationship to enzymes of the glycogen synthase kinase-3 category

A heparin-activated protein kinase has been previously identified in rabbit skeletal muscle extracts (Z. Ahmad et al. (1985) FEBS Lett. 179, 96-100). Further study has indicated that this enzyme phosphorylates rabbit muscle glycogen synthase in the same tryptic peptide(s) as the protein kinase FA/GSK-3 (glycogen synthase kinase-3) and is able to activate the ATP-Mg2+-dependent protein phosphatase. These results indicate similarities in properties between the two protein kinases. Exposure of the heparin-activated enzyme to trypsin resulted in loss of heparin activation, from 3-fold to 1.3-fold. One hypothesis suggested by this result is that the enzyme FA/GSK-3 could be a derivative of the heparin-activated enzyme that has lost heparin sensitivity. The conceptual importance of this hypothesis is that it may provide a clue to the mode of regulation of this important class of protein kinases.     

Regulation of the activity of rabbit skeletal muscle phosphorylase kinase by glycogen

The effects of glycogen on the non-activated and activated forms of phosphorylase kinase were studied. It was found that in the presence of glycogen the activity of non-activated kinase at pH 6.8 and 8.2 and that of the activated (in the course of phosphorylation) form are enhanced. The degree of activation depends on glycogen concentration. At saturating concentrations, this enzyme activity increases 2-3-fold; the enzyme affinity for the protein substrate, phosphorylase b, also shows an increase. The polysaccharide has no effect on the activity of phosphorylase kinase stimulated by limited proteolysis. In the presence of glycogen, the rate of autocatalytic phosphorylation of the enzyme is increased. Glycogen stabilizes the enzyme activity upon dilution. The experimental results suggest that the polysaccharide directly affects the phosphorylase kinase molecule. The maximal binding was shown to occur at the enzyme/polysaccharide ratio of 1:10 (w/w) in the presence of Ca2+ and Mg2+.     

Donnan potentials in rabbit psoas muscle in rigor.

Collins and Edwards (1971, Am. J. Physiol., 221:1130-1133) have shown that a tissue potential can be measured with microelectrodes in glycerinated muscle and that this potential is consistent with a Donnan equilibrium of small ions due to the concentration of net fixed electric charge on the contractile proteins. This approach has been combined with x-ray and light diffraction measurements of the muscle lattice dimensions, and the data are used to determine the thick filament charge and thin filament charge under a variety of different conditions. The thick filament charge is a function of the bathing solution, in particular its pH and ionic composition. These parameters are important in determining the volume of the equilibrium lattice and possibly are involved in the contraction mechanism itself.     

Tension responses to increased hydrostatic pressure in glycerinated rabbit psoas muscle fibres

A method developed to study the effect of increased hydrostatic pressure on the isometric tension of a single muscle fibre is described and experiments done at room temperature (18-22 degrees C) on glycerinated rabbit psoas muscle fibres are presented. Increase of pressure (range 1-10 MPa) caused little change in tension transducer response when a muscle fibre was relaxed. However, there was a reversible depression of isometric tension with an increase of pressure when a fibre was maximally calcium-activated or in rigor; the depression was around 15% for active tension and 30% for rigor tension, for an increase of pressure of 10 MPa (ca. 100 atm).     

Binding and location of AMP deaminase in rabbit psoas muscle myofibrils

It is shown that an interaction exists between AMP deaminase (EC 3.5.4.6) and myofibrils that is sufficiently strong (Kd congruent to 10(-10) M) for more than 99% of the binding sites for the enzyme to be filled in vivo. The binding is not strong enough, however, to stop removal of the enzyme during the extensive washing normally used in the preparation of myofibrils. Fluorescent antibodies to the enzyme label myofibrils close to the junction of the A- and I-bands. The invariance of the position of the antibody stripes at this site, over a range of sarcomere lengths, indicates that the enzyme is attached to the A-band. The intensity of the fluorescence declines in parallel with dissociation of the enzyme. In this muscle, the number of AMP deaminase binding sites per thick filament is approximately six, suggesting that the enzyme is located at a single axial position in each half A-band. Electron microscopy of negatively stained, antibody-labelled myofibrils reveals the distance between the AMP deaminase sites at opposite ends of an A-band to be 1.69(+/- 0.02 micron). Since the length of the A-band is 1.57 micron, the binding site for the enzyme must be significantly beyond where thick filaments have previously been thought to end.     

Identification of binding sites on protein targeting to glycogen for enzymes of glycogen metabolism

The activation of protein phosphastase-1 (PP1) by insulin plays a critical role in the regulation of glycogen metabolism. PTG is a PP1 glycogen-targeting protein, which also binds the PP1 substrates glycogen synthase, glycogen phosphorylase, and phosphorylase kinase (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Through a combination of deletion analysis and site-directed mutagenesis, the regions on PTG responsible for binding PP1 and its substrates have been delineated. Mutagenesis of Val-62 and Phe-64 in the highly conserved (K/R)VXF PP1-binding motif to alanine was sufficient to ablate PP1 binding to PTG. Phosphorylase kinase, glycogen synthase, and phosphorylase binding all mapped to the same C-terminal region of PTG. Mutagenesis of Asp-225 and Glu-228 to alanine completely blocked the interaction between PTG and these three enzymes, without affecting PP1 binding. Disruption of either PP1 or substrate binding to PTG blocked the stimulation of PP1 activity in vitro against phosphorylase, indicating that both binding sites may be important in PTG action. Transient overexpression of wild-type PTG in Chinese hamster ovary cells overexpressing the insulin receptor caused a 50-fold increase in glycogen levels. Expression of PTG mutants that do not bind PP1 had no effect on glycogen accumulation, indicating that PP1 targeting is essential for PTG function. Likewise, expression of the PTG mutants that do not bind PP1 substrates did not increase glycogen levels, indicating that PP1 targeting glycogen is not sufficient for the metabolic effects of PTG. These results cumulatively demonstrate that PTG serves as a molecular scaffold, allowing PP1 to recognize its substrates at the glycogen particle.     

Mechano-chemical properties of actomyosin-ADP complexes in rabbit psoas muscle fibers

The relaxing effect of vanadate on active contractile system is found to be completely absent from rigor skinned fibres with ADP even on their stretching up to the forces comparable with the active ones, though vanadate is likely to bind not very firmly with crossbridges not containing inorganic phosphate. Probable reasons of such distinction are considered. The complex actomyosin-ADP in the rigor fibres is supposed to have significantly lower free energy independently of its deformation than the one of the same composition in the active ones. Possible role of different actomyosin-ADP states in the mechanochemical cycle of crossbridge is discussed.     

X-ray diffraction study of the rabbit psoas muscle with extracted H-zone

Fibre bundles of glycerinated rabbit psoas muscle about 0.5-1.0 mm thick were incubated in 5 mM tris-(hydroxymethyl)-aminomethane (Tris), pH 8.0 for 5-20 hours at 4 degrees C. This treatment leads to selective removal of some proteins in the M-bands and H-zones of sarcomeres. Effects of extraction were analyzed on the basis of electron micrographs of longitudinal sections of muscle specimens, gel electrophoresis patterns of myofibrils and of the extracts, and measurements of the creatine kinase activity of myofibrils. In the X-ray diffraction patterns of the fibre bundles subjected to prolonged extraction a drastic decrease in the intensity of "442 A" and "223 A" meridional reflections and a considerably smaller decrease in the intensity of "212 A" meridional reflections were observed. The "147 A" meridional reflection remains practically unchanged. It was concluded that: (1) The reflections "442 A" and "223 A" were contributed mainly by diffraction on the minor proteins located in the central part of the thick filaments in between the C-zones. This is contrary to the widely accepted viewpoint according to which the appearance of "442 A" reflection is caused only by the C-protein component of the thick filaments. (2) The "147 A" meridional reflection is contributed mainly by C-protein and light meromyosin of the thick filaments.     

Effect of limited proteolysis on activity and phosphorylation of rabbit muscle glycogen synthetase.

Purified rabbit skeletal muscle glycogen synthetase, in both the glucose-6-phosphate (P)-dependent (phosphorylated) and the glucose-6-P-independent (dephosphorylated) forms, was subjected to limited proteolysis by trypsin. Both forms could be degraded from their original subunit molecular weight of 85,000 to 76,000 and subsequently to 68,000, as determined with acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Degradation of the glucose-6-P-dependent form of the enzyme resulted in essentially no change in the activity when measured either in the presence or in the absence of glucose-6-P. Degradation of the glucose-6-P-independent form was associated with a progressive increase in glucose-6-P dependency. Phosphorylation of the glucose-6-P-independent form with the adenosine 3′,5′-monophosphate-dependent protein kinase and subsequent digestion of the ³²P-labeled enzyme showed that the phosphate group was retained on these subunits. The protein kinase phosphorylated both the original subunit with molecular weight 85,000 and the partially digested subunit with molecular weight 76,000. Upon further digestion of the enzyme into a form having a subunit molecular weight of 68,000, the enzyme was unable to accept a phosphate group from ATP. By contrast with the phosphorylation reaction, the dephosphorylation reaction catalyzed by partially purified glycogen synthetase phosphatase is not stringent in terms of structural integrity of the synthetase. The phosphatase dephosphorylated the glucose-6-P-dependent form of glycogen synthetase equally well at various degrees of degradation.