Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.     

Studies on phosphorylase isozymes in lower vertebrates. Purification and properties of lamprey phosphorylase.

Skeletal muscle phosphorylase b has been purified from lamprey, Entosphenus japonicus, to a state of homogeneity as judged by the criterion of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme was completely dependent on AMP for activity and converted into the a form by rabbit muscle phosphorylase kinase in the presence of ATP and Mg²⁺. The subunit molecular weight determined by SDS-gel electrophoresis was 94,000 ± 1,600 (SE). The enzyme activity was stimulated by Na₂SO₄, but was not affected by mercaptoethanol. The K_m values of the a form for glucose 1-phosphate and glycogen were 3.5 mm and 0.13%, respectively, and those of the b form for glucose 1-phosphate, glycogen, and AMP were 15 mm, 0.4%, and 0.1 mm, respectively. These values were smaller than those reported with lobster phosphorylase and greater than those reported with mammalian skeletal muscle phosphorylases. Electrophoretic and immunological studies have indicated that lamprey phosphorylase b exists as a single molecular form in skeletal muscle, heart, brain, and kidney. Rabbit antibody against lamprey phosphorylase cross-reacted with phosphorylases from skate and shark livers more intensely than with those from skeletal muscles.     

Muscle glycogenolysis. Regulation of the cyclic interconversion of phosphorylase a and phosphorylase b

Regulation of glycogenolysis in skeletal muscle is dependent on a network of interacting enzymes and effectors that determine the relative activity of the enzyme phosphorylase. That enzyme is activated by phosphorylase kinase and inactivated by protein phosphatase-1 in a cyclic process of covalent modification. We present evidence that the cyclic interconversion is subject to zero-order ultrasensitivity, and the effect is responsible for the "flash" activation of phosphorylase by Ca2+ in the presence of glycogen. The zero-order effect is observable either by varying the amounts of kinase and phosphatase or by modifying the ratio of their activities by a physiological effector, protein phosphatase inhibitor-2. The sensitivity of the system is enhanced in the presence of the phosphorylase limit dextrin of glycogen which lowers the Km of phosphorylase kinase for phosphorylase. The in vitro experimental results are examined in terms of physiological conditions in muscle, and it is shown that zero-order ultrasensitivity would be more pronounced under the highly compartmentalized conditions found in that tissue. The sensitivity of this system to effector changes is much greater than that found for allosteric enzymes. Furthermore, the sensitivity enhancement increases more rapidly than energy consumption (ATP) as the phosphorylase concentration increases. Energy effectiveness is shown to be a possible evolutionary factor in favor of the development of zero-order ultrasensitivity in compartmentalized systems.     

Regulation of platelet phosphorylase.

A sensitive fluorimetric enzyme assay was developed for study of activation of glycogen phosphorylase (EC 2.4.1.1) in intact platelets and in platelet extracts. Activity was calculated as AMP independent (activity in the absence of AMP), total (activity in the presence of 1 mM AMP), and AMP dependent (difference between AMP independent and total). The following observations were made with intact rat platelets. (1) Stimulation of platelets with thrombin caused a 7-fold increase in total activity, with increases in both AMP-dependent and AMP-independent activities. Maximum activation was obtained within 10 s after addition of thrombin. (2) The divalent cation ionophore A23187 caused a similar, though less pronounced, activation of phosphorylase. (3) Acceleration of glycogenolysis by inhibition of respiration with cyanide caused similar changes in phosphorylase activity but with the maximum effect observed only after 45 s. (4) Dibutyryl cyclic AMP had two effects; it partially activated phosphorylase and blocked further activation by thrombin, but not A23187. Similar effects were observed with human platelets, but low resting levels of phosphorylase activity could not be maintained so that changes were not as large as with rat platelets. Experiments with extracts of rat platelets gave the following results. (1) Phosphorylase activity in many extracts of non-stimulated platelets could be increased by incubation with Mg2+-ATP and Ca2+; ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) partially inhibited. (2) In some extracts there was essentially no activation by incubation with Mg2+-ATP and Ca2+, but addition of cyclic AMP GAVE PARTIAL ACTIVATIon while addition of rabbit muscle phosphorylase kinase gave full activation. (3) Incubation of extracts of thrombin-stimulated platelets caused conversion of AMP-dependent to AMP-indeptndent activity. It is concluded that platelet phosphorylase exists in an inactive and two active forms. Conversion of the inactive to the active forms and of the AMP-dependent to the AMP-independent form is catalyzed by a kinase(s) that requires Ca2+ for full activity and is activated through a cyclic AMP-mediated process. The major change following physiological stimulation is an increase in both active forms, with little change in their ratio.     

Isozymes of phosphorylase kinase in rabbit skeletal muscle. Functional implications of differences in phosphorylase kinase and phosphorylase activities in individual muscle fibers

Immunological and microanalytical methods were used to investigate the two isozymes of phosphorylase kinase, enzyme w and enzyme r, in psoas major and tibialis anterior muscles. Peptide mapping experiments indicated that the alpha subunit of enzyme w and alpha' subunit of enzyme r were structurally very similar. Both subunits were completely immunoprecipitated from muscle extracts with an antibody specific for the beta subunit of the kinase, indicating that alpha and alpha' subunits are completely assembled with beta subunits in adult muscle fibers. The relative amounts of enzymes w and r in single fibers were determined from amounts of alpha and alpha' subunits, which were detected by immunoblotting. Phosphorylase kinase and phosphorylase activities were measured in the same fibers, as well as in individual fibers from diaphragm and soleus muscles. Slow oxidative fibers were found to contain low levels of enzyme r, but almost no enzyme w. Considerably more enzyme r was present in fast oxidative-glycolytic fibers. Fast glycolytic fibers contained the most enzyme w, and the highest levels of enzyme r were found in a subgroup of such fibers. Interestingly, more than half of the fast glycolytic fibers analyzed contained both isozymes. In these fibers phosphorylase was positively correlated with enzyme w, but negatively correlated with enzyme r. Total kinase activity ranged 30-fold from the highest in one of the psoas fibers to the lowest in one of the soleus fibers and was closely correlated with the phosphorylase levels. In psoas and soleus fibers, calculated absolute maximal rates for phosphorylase b to a conversion varied almost 2,500-fold.     

Electrophoretic studies on the phosphorylase isozymes.

The electrophoretic method of Davis, Schliselfeld, Wolf, Leavitt and Krebs (1967) for phosphorylase isozymes has been modified. By this method, five isozymes were separated in various organs of rat and pig and were disignated as phosphorylase L, LI, I, II and III. The L and III enzymes were the only forms found in liver and skeletal muscle, respectively, while the I enzyme was dominant in brain, uterus, lung and small intestine, which also contained some fractions of the II and III enzymes. The I enzyme was also dominant in adrenal, ovary and kidney, but these organs contained the L+II or L+LI as minor components. The L and LI were richly found in spleen and leukocytes of adult rats and pigs and in liver of newborn rats. Such organ-specific heterogeneity of phosphorylase was confirmed by the immunological tests with the antibodies prepared against phosphorylases I, III and L. The II and LI enzymes were found to be the hybrid molecules between the I and III enzymes, and between the I and L enzymes which have been previously reported as unhybridizable, respectively. In view of the above findings, it was concluded that the rat and pig possessed at least five molecular forms of phosphorylase.     

Hydrocarbon-coated sepharoses. Use in the purification of glycogen phosphorylase

A homologous series of hydrocarbon-coated Sepharoses varying in the length of their alkyl side chains (Seph-NH(CH₂)_nH) was synthesized. These modified Sepharoses provide a versatile tool for the purification of proteins, since, by choosing the suitable member of the series, a desired protein can be extracted from a protein mixture. This is illustrated in the case of glycogen phosphorylase, which is not retained at all by methyl Sepharose (n=1), is retarded by propyl Sepharose (n=3), is adsorbed on butyl Sepharose (n=4) and can be eluted from the column by deforming buffers, and is so tightly adsorbed on hexyl Sepharose (n=6) that it could be eluted from the column only in the denatured form, by washing with 0.2 N CH₃COOH. On a preparative scale, a hundred-fold purification of phosphorylase could be achieved in one step, by passage of a crude muscle extract on a small butyl Sepharose column.     

Subunit interactions and the allosteric response in phosphorylase.

The contribution of intersubunit interactions to allosterically induced conformational changes in phosphorylase are considered. Phosphorylase a, Pa (phosphorylated at Ser-14), is significantly in the active (R) conformation, while phosphorylase b, Pb (nonphosphorylated), is predominantly in the inactive (T) conformation. The structure of glucose-inhibited (T) Pa has been determined at 2.5-A resolution and atomic coordinates have been measured. These data have been used to calculate the solvent accessible surface area at the subunit interface and map noncovalent interactions between protomers. The subunit contact involves only 6% of the Pa monomer surface, but withdraws an area of 4,600 A2 from solvent. The contact region is confined to the N-terminal (regulatory) domain of the subunit. Half of the residues involved are among the 70 N-terminal peptides. A total of approximately 100 atoms take part in polar or nonpolar contacts of less than 4.0 A with atoms of the symmetry-related monomer. The contact surface surrounds a central cavity at the core of the interface of sufficient volume to accommodate 150-180 solvent molecules. There are four intersubunit salt bridges. Two of these (Arg 10/Asp 32, Ser-14-P/Arg 43) are interactions between the N-terminus of one protomer with an alpha-helix loop segment near the N-terminus of the symmetry-related molecule. These two are relatively solvent accessible. The remainder (Arg 49/Glu 195, Arg 184/Asp 251) are nearer the interface core and are less accessible. The salt bridges at the N-terminus are surrounded by the polar and nonpolar contacts which may contribute to their stability. Analysis of the difference electron density between the isomorphous Pa and Pb crystal structures reveals that the N-terminal 17 residues of Pb are disordered. Pb thus lacks two intermolecular and one intersubunit (Ser-14-P/Arg 69) salt linkage present in Pa. The absence of these interactions in Pb is manifested in the difference in the free energy of T leads to R activation, which is 4 kcal more than that for Pa. Difference Fourier analysis of the T leads to R transition in substrate-activated crystals of Pa suggests that the 70 N-terminal residues undergo a concerted shift towards the molecular core; salt bridges are probably conserved in the transition. It is proposed that the N-terminus, when "activated" by phosphorylation (via a specific kinase) behaves as an intramolecular "effector" of the R state in phosphorylase and serves as the vehicle of homotropic cooperativity between subunits of the dimer.     

Phosphorylation of synthetic peptide analogs of the phosphorylatable site of phosphorylase b with phosphorylase kinase.

A synthetic octapeptide of the phosphorylatable site of phosphorylase and its analogs were used to determine the specificity of nonactivated phosphorylase kinase. By substitution of each of six amino acid residues (lysine₁₁, glutamine₁₂, isoleucine₁₃, serine₁₄, valine₁₅, and arginine₁₆), it was found that these residues were all important in the enzyme recognition. Valine₁₅ was more important than isoleucine₁₃, when either valine₁₅ or isoleucine₁₃ was substituted by glutamic acid. A peptide containing two isoleucyl residues (surrounding serine₁₄) had a better phosphorylation rate than a peptide containing two valyl residues. A peptide with a threonine residue instead of serine could be phosphorylated but with a low reaction rate.