Purification and properties of polycation-stimulated phosphorylase phosphatases from rabbit skeletal muscle

Four types of polycation-stimulated (PCS) phosphorylase phosphatases have been isolated from rabbit skeletal muscle. They are called PCSH (390 kDa), PCSM (250 kDa), and PCSL (200 kDa) phosphatase according to the apparent molecular weight of the native enzymes in gel filtration. Two forms of PCSH phosphatase could be separated by Mono Q fast protein liquid chromatography: PCSH1 and PCSH2. In the absence of polycations, the specific activities of the PCSH1, PCSH2, PCSM, and PCSL phosphatase were 400, 680, 600, and 3000 units/mg, respectively, using phosphorylase a as a substrate. They all contain a 62-65- and a 35-kDa subunit, the latter being the catalytic subunit. In addition PCSH1 phosphatase contains a 55-kDa subunit and the PCSM phosphatase a 72-75-kDa subunit in a substoichiometric ratio. All the PCS phosphatases are insensitive to Ca2+ calmodulin, inhibitor-1, and modulator protein. They display a high specificity for the alpha-subunit of phosphorylase kinase and a broad substrate specificity. The PCSH1 and PCSH2 phosphatases, but not the catalytic subunit (PCSC phosphatase), show a high degree of specificity for the deinhibitor protein. During the purification the phosphorylase to inhibitor-1 phosphatase activity ratio (10:1) remained constant for the PCSH and PCSL enzymes but decreased for the PCSM phosphatase. The stimulation observed with low concentrations of polycations is enzyme directed. The different enzyme forms show a characteristic concentration optimum and degree of stimulation. At higher concentrations, polycations become inhibitory and a time-dependent deactivation of the phosphatases is observed.     

Purification, quaternary structure and immunological properties of phosphorylase kinase from chicken skeletal muscle

Phosphorylase kinase was isolated from red and white chicken skeletal muscle in a nearly homogeneous state as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme as determined by gel filtration on Sepharose 4B is close to that of rabbit skeletal muscle phosphorylase kinase (i. e., approximately 1300 000). The molecular weights of the subunits determined by SDS gel electrophoresis are: alpha', 140 000 beta, 129 000; gamma', 44 000; delta, 17 000 (cf. the Mr values of the alpha- and gamma-subunits of the rabbit muscle isoenzyme are 146 000 and 42 000). The four subunits, alpha', beta, gamma' and delta, were found to exist in equimolar amounts as shown by a densitometric analysis of acrylamide gels; hence, the subunit formula of the chicken skeletal muscle isoenzyme is (alpha' beta gamma' delta)4. Rabbit antisera against a mixture of alpha'- and beta-subunits of chicken phosphorylase kinase yield a single precipitin line with this enzyme, do not show cross reactions of identity with the rabbit muscle enzyme but strongly inhibit the activity of the chicken enzyme and partially inhibit the activity of the rabbit muscle isoenzyme.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Purification of glycogen phosphorylase from small quantities of mouse skeletal muscle

A new approach to the purification of skeletal muscle glycogen phosphorylase is described. The purification scheme is particularly suited to preparation of the enzyme from small amounts of tissue. A combination of dye-ligand chromatography and hydrophobic chromatography yields homogenous enzyme with good recoveries. The purification is rapid and may be completed in a working day.     

Purification and properties of glycogen phosphorylase isolated from bovine skeletal muscles

Glycogen phosphorylase isolated from bovine skeletal muscles was found to be homogeneous during polyacrylamide gel electrophoresis. The enzyme phosphorylation by phosphorylase kinase is accompanied by the incorporation of one mole of labeled phosphate per protein dimer; therefore the enzyme is represented by a partly phosphorylated form. The presence of a phosphate group prevents the removal of the protein-bound pyridoxal phosphate. The partly phosphorylated bovine phosphorylase possesses a low affinity for AMP and is inactive in the presence of IMP. Bovine phosphorylase a obtained from the partly phosphorylated enzyme has a molecular mass corresponding to a dimer. Both forms of bovine phosphorylase exhibit high cooperativity towards the substrate. The mechanism of phosphorylase a activation by AMP and IMP is identical: the nucleotides increase the enzyme affinity for the substrate as well as the maximal rate of the enzymatic reaction. Study of the enzyme inhibition by caffeine revealed the cooperativity of caffeine-binding centers. The equilibrium between the active and inactive enzyme conformations in the presence of caffeine is markedly shifted towards the inactive (T) form of glycogen phosphorylase.     

Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation

Transverse tubule vesicles were isolated from frog skeletal muscle by a procedure initially described by Rosemblatt et al. (J. Biol. Chem. 256, 8140-8148 (1981)) and later modified by Hidalgo et al. (J. Biol. Chem. 258, 13937-13945 (1983]. A large fraction of the isolated vesicles (80-90%) were sealed, as indicated by the detergent induced increase in (Na+ + K+)-ATPase activity and ATP-dependent ouabain binding. To determine the orientation of the sealed vesicles binding of digoxin, a lipid soluble derivative of ouabain, was measured. The same values of ATP-dependent digoxin binding were found with or without detergents, indicating that all the vesicles that are sealed have the ATP site accessible, and hence are sealed with the cytoplasmic side-out (inside-out orientation). The transverse tubule preparation isolated from frog muscle is highly purified, as indicated by its cholesterol content and its (Na+ + K+)-ATPase activity; negligible contamination with sarcoplasmic reticulum was observed, as indicated by the protein composition and the lack of measurable Ca2+-ATPase activity of the isolated transverse tubules. High initial rates of Mg2+-ATPase activity were found, with the peculiar property of being inhibited during the course of the reaction. Addition of lysophosphatidylcholine or saponin partially prevented the inhibition of Mg2+-ATPase activity during the reaction.     

Elastic and viscous properties of resting frog skeletal muscle.

The mechanical properties of the resting, whole semitendinosus muscle of the frog have been characterized as functions of both muscle length and temperature. Measurements were made of pseudorandom white noise (PRWN) displacements (less than 10 A/half-sarcomere) applied to the muscle and the force responses to these movements. Signal correlation techniques were then used to obtain the dynamic modulus function for the muscle in the frequency range 2.44-320 Hz. This function was represented by a series combination of a Voigt element and a time delay element for tension propagation along the muscle. A dynamic elastic modulus (E), coefficient of damping (B), and tension transmission velocity (V) were measured for resting muscle on the basis of this model. For each of these parameters, a marked variation with sarcomere length (s) was found. The mean values for E and B at LO (s=2.25 mum) were 1.84+/-0.24 X 10(5) N/m2 and 2.33+/-0.25 X 10(2) Ns/m2, respectively. Further, B demonstrated a negative temperature dependence, Q10=0.78 (P less than 0.05), in the range s=2.6-3.0 mum, while E was not significantly temperature dependent. The length-dependent variations of E and B are interpreted as deriving from both passive muscle elements and attached crossbridges. Velocity was calculated at a single displacing frequency for every experiment; the mean value at LO and all temperatures was v=11.7+/-0.6 m/s. Velocity was also calculated as a function of frequency within several experiments: the results indicate considerable variation of v with frequency.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Bovine skeletal muscle adenosine deaminase. Purification and some properties

A low molecular weight form of adenosine deaminase from bovine skeletal muscle was purified about 930-fold. The enzyme had a mol. wt of 31,000, a Km value for adenosine of 2.37 X 10(-5) M and a pH optimum at 7.0. This enzyme is very resistant to heat inactivation and does not require metal activators or other dialysable cofactors. A possible role in the post-mortem metabolism of adenine nucleotide in skeletal muscle is discussed.     

Purification and properties of aminopeptidase H from chicken skeletal muscle.

Aminopeptidase H was purified from fresh chicken breast muscle by ammonium sulfate fractionation and successive chromatographies on DEAE-cellulose, Ultrogel AcA 34, activated thiol-Sepharose 4B, phenyl-Sepharose CL-4B and DEAE-cellulose again. The purified enzyme migrated as a single band on SDS/PAGE. Aminopeptidase H exhibits activity against both L-leucine beta-naphthylamide and alpha-N-benzoyl-DL-arginine beta-naphthylamide. The molecular mass of this enzyme was found to be 52 kDa on SDS/PAGE and 400 kDa on Sepharose 6B column chromatography. The optimum pH for the hydrolysis of both substrates was 8.0 and this activity was remarkably enhanced by reducing agents. The enzyme was strongly inhibited by monoiodoacetate and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, bestatin or puromycin. Aminopeptidase H has been shown to hydrolyze di-, tri- and tetrapeptides in the manner of an aminopeptidase, as well as the beta-naphthylamide derivatives of amino acids. However, the enzyme has not been shown to hydrolyze proteins such as hemoglobin, bovine serum albumin, myofibrillar proteins or sarcoplasmic proteins.     

Purification and some properties of cathepsin H from rabbit skeletal muscle

Rabbit muscle cathepsin H classified as an aminoendopeptidase was purified and its properties were investigated to clarify its contribution to the proteolysis of postmortem muscle. The purification was performed by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocelluose, DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme gave a single protein band on SDS-PAGE. Its molecular mass was found to be 28 kDa by gel permeation and 30 kDa by SDS-PAGE. The optimum pHs for alpha-N-benzoyl-DL-arginine-beta-naphthylamide (BANA)- and L-leucine-beta-naphthylamide (Leu-NA)-hydrolyzing activities were 6.6 and 7.0, respectively. This enzyme was almost stable in the range of pH 4-5 and up to 50 degrees C at pH 5.0. The Km values of BANA- and Leu-NA-hydrolyzing activities were 0.367 and 0.203 mM, respectively. The enzyme was inhibited by monoiodoacetic acid, antipain, leupeptin, TLCK and TPCK, but was not affected by pepstatin, bestatin, puromycin, PMSF or trypsin inhibitor. This enzyme strongly acted on Arg-, Lys-, Met-, Ala-, Ser- and Leu-NAs, weakly acted on Val- and Glu-NAs, and hardly acted on Pro- and Gly-NAs. The amount of cathepsin H in muscle was estimated to be less than one-fourth of the sum of the amount of aminopeptidases C and H by the Leu-NA-hydrolyzing activity on the chromatography. This enzyme degraded myosin heavy chain, actin, tropomyosin and troponin I clearly at pH 4.0, while it slightly degraded troponin I at pH 5.0-5.6. Therefore, the contribution of cathepsin H to the proteolysis of postmortem muscle is presumed to be relatively small.     

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.     

Purification and properties of cytosolic malic enzyme from human skeletal muscle

1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme.     

Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle

Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.