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L M Heilmeyer

Publications and source records attributed to L M Heilmeyer.

At least 73 records · Page 4Linked to original sources
PubMed1984

Sequential phosphorylation of skeletal muscle troponin.

Phosphorylation of the isolated rabbit skeletal muscle holotroponin complex at troponin-T by phosphorylase kinase is unusual in that it shows maxima and minima. These oscillations are due to protein phosphatase activity present in the preparations. Following tryptic digestion two phosphorylated peptides, I and II, can be isolated. Their amino-acid compositions are identical and correspond to that of the tryptic peptide which contains the two known phosphorylatable sites 149/150 and 156/7 of troponin-T. Peptide I is phosphorylated on both sites and peptide II only on one site. During phosphorylation the doubly phosphorylated peptide I appears first; after a short lag phase peptide II is formed containing only one phosphate. These phenomena probably cause the observed oscillations in the degree of the holotroponin phosphorylation.

Animals↗
PubMed1983

Shape and substructure of skeletal muscle myosin light chain kinase.

To evaluate the shape and substructure of calmodulin-dependent myosin light chain kinase from skeletal muscle, the apo- and holoenzyme and three well-characterized proteolytic fragments were studied by enzymatic measurements, by hydrodynamic techniques, and by CD spectroscopy. For the native apoenzyme, a molecular weight of 70 300 was established by sedimentation equilibrium in contrast to greater than 80 000 estimated by electrophoresis. A highly asymmetric structure was evidenced from sedimentation and viscosity data. Examination of two slightly different calmodulin binding fragments of Mr approximately 36 000 showed that both are fairly globular, high in alpha-helix content, enzymatically active, and calmodulin regulated. They have been termed head fragments. The third fragment of Mr approximately 33 000 could be demonstrated to represent the remaining part of the native enzyme by its amino acid composition and CD spectrum. This enzymatically inactive fragment, although low in alpha-helix content and rich in proline, was shown to be highly asymmetric (a/b greater than 10). From the latter, termed tail fragment and one of the head fragments, a more active enzyme could be partially reconstituted. Modeling by spherical beads [Bloomfield, V., Dalton, W. O., & Van Holde, K. E. (1967) Biopolymers 5, 135-148] led to a close agreement in observed and calculated frictional ratios for all fragments as well as the apoenzyme built up by end to end arrangement of head and tail fragment, suggesting this headed structure for the enzyme. Holoenzyme formation by calmodulin binding to the head was accompanied by an increase in asymmetry and alpha-helix content and a decrease in apparent partial specific volume.

Amino Acids↗
PubMed1983

Phosphofructokinase is a calmodulin binding protein.

A trial to purify myosin light chain kinase from crude myosin led to the isolation of a Mr 85 000 calmodulin binding protein different from this enzyme. Because it showed inherent phosphofructokinase activity we investigated its relation to this enzyme. We demonstrated identity to phosphofructokinase by a close to identical amino acid composition, by antigenic identity and a set of completely identical peptide maps. The calmodulin binding property was also shown for a fraction of the enzyme prepared by standard methods. First experiments show that Ca2+--calmodulin is a potent regulator of phosphofructokinase polymerization.

Amino Acids↗
PubMed1983

Skeletal muscle myosin light chain kinase. A refined structural model.

A hydrodynamic, enzymatic and CD spectroscopic study of skeletal muscle myosin light chain kinase, three proteolytic fragments and corresponding complexes with calmodulin was performed. A refined shape model was built for the enzyme. It was shown that a head-and-tail structure is formed from two major fragments which are aligned end-to-end. The one fragment (Mr 36000) is compact, of high alpha-helix content and contains the catalytic center with the light chain and the calmodulin binding domains. The other fragment (Mr 33000) with unknown function is asymmetric (a/b greater than 10), of low alpha-helix and of unusually high proline content.

Animals↗
PubMed1983

Ca2+ and Mg2+-dependent complex formation of tropomyosin with phosphotroponin (P1TI2C) or dephosphotroponin (TI2C).

The reduced viscosity of troponin and dephosphotroponin is independent of the protein concentration in both states, either metal-free or with troponin C saturated with Ca2+ or Mg2+; that of tropomyosin increases linearly as function of the protein concentration, indicating aggregation. Addition of troponin to tropomyosin increases the reduced viscosity over the expected value being maximal at a 1:1 molar ratio of both proteins. The reduced viscosity of a 1:1 molar mixture of phosphotroponin-Mg4 or dephosphotroponin-Mg3 increases in two phases as function of the total protein concentration, indicating the formation of two kinds of troponin-tropomyosin complexes. In the first phase, troponin and tropomyosin form a non-aggregating 1:1 complex, which is characterized by a value of 0.45 dl/g for the intrinsic viscosity and a sedimentation coefficient of 3.6 S. Employing these two values a molecular weight of 150 000 can be calculated, which is in the range of the sum of molecular weights for troponin and tropomyosin (156 000). In the second phase the troponin-tropomyosin complex aggregates further, a process described by:n (troponin-tropomyosin) leads to (troponin-tropomyosin)n. This further aggregation occurs upon saturation of the Ca2+-specific sites in troponin C. A model is discussed which explains the shortening of 1.5 nm per tropomyosin molecule upon the shift of tropomyosin from the periphery into the groove of the actin filament by tropomyosin aggregation.

Animals↗
PubMed1982

Multiple activities on phosphorylase kinase. 2. Different specificities toward the protein substrates phosphorylase b, troponin, and phosphorylase kinase.

Phosphorylase kinase exhibits three kinds of enzymatic activities. A partial activity, A0, catalyzes the phosphorylation of phosphorylase b, troponin I, and phosphorylase kinase itself (autophosphorylation); A1 can utilize only phosphorylase b and phosphorylase kinase as the substrate, whereas A2 can utilize only phosphorylase b and troponin T. Stimulation of A1 by Ca2+ coincides with an increase in the number of sites that can undergo self-phosphorylation ranging from ca. 35 to ca. 70 mol of phosphate incorporated/1.28 X 10(6) g of proteins. Inhibition of A0 and A1 by millimolar Ca2+ is accompanied by a decrease in substrate availability during self-phosphorylation. NH4Cl (150 mM) strongly inhibits the availability of troponin as a substrate. In the course of self-phosphorylation, the activities A0 and A1 are both stimulated moderately by an increase in pH; however, only A1 shows some inhibition by 150 mM NH4Cl. Millimolar Ca2+ inhibits A1 and A2 as measured by self-phosphorylation or troponin phosphorylation, as observed with the phosphorylation of phosphorylase b [Kilimann, M. W., & Heilmeyer, L. M. G., Jr. (1982) Biochemistry (preceding paper in this issue)]. The rate of self-phosphorylation varies as a function of substrate concentration (Km = 68 nM at 10 mM Mg2+ and 184 microM Ca2+, pH 9.0). The data indicate that both Ca2+ activation and inhibition seem to be mediated by phosphorylase kinase itself rather than by the substrates.

Ammonium Chloride↗
PubMed1981

The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle.

Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

X-linked dominant inheritance of partial phosphorylase kinase deficiency in mice.

A new mouse strain, the V strain, with a partial deficiency of phosphorylase kinase has been established. The deficiency is caused by an X-linked dominant gene (PhKc). Muscle extracts of homozygous and heterozygous females and hemizygous males have about 25% of the activity found in extracts of normal (C3H/HeHan) mice. This dominant phosphorylase kinase deficiency of the new V strain is different from that of the I-strain mice with the X-linked recessive deficiency of skeletal muscle phosphorylase kinase. The muscle extracts of V-strain and normal mice contain the same phosphorylase phosphatase activity of about 1 U/mg. Heart and liver extracts from V mice contained about 50% and 66%, respectively, of the phosphorylase kinase activity compared to that found in the same organs from the normal mice. The glycogen content of the skeletal muscle of the V strain was normal, i.e., 0.9 mg/g. Phosphorylase kinase was purified from the skeletal muscle of the V strain by (a) hydrophobic chromatography on methylamine Sepharose, (b) ammonium sulfate precipitation, and (c) gel filtration of Sepharose 4B. The enzyme has a similar structure to the normal murine and rabbit skeletal muscle enzyme, except that the proportion of the subunits differs. The molar ratio of the subunits of the V strain mice is (alpha + alpha'):beta:gamma=0.54:1:1.169, in comparison with that of the rabbit (alpha + alpha'):beta:gamma=1.1:1.0:1.0 and that of normal murine enzyme 0.9:1.0:0.7.

Animals↗
PubMed1980

Comparison of the Mg2+ and Ca2+ binding properties of troponin complexes P1-TI2C and TI2C.

The phosphoserine present in troponin T of freshly isolated skeletal muscle troponin P1-TI2C was dephosphorylated by alkaline phosphatase and the resulting troponin TI2C characterized by phosphorous content and gel electrophoresis in presence of sodium dodecylsulfate. Both complexes bind Ca2+ in an identical manner with a K0.5 of 5.3 X 10(-9) M for the Ca2+/Mg2+ binding sites and of 1.1 X 10(-6) M for the Ca2+-specific sites. 3.5 mM Mg2+ lowers the K0.5 value at the Ca2+/Mg2+ binding sites of 1.3 X 10(-7) M in the phospho-troponin P1-TI2C and leaves nearly unchanged the value of the dephosphorylated troponin TI2C at 1.2 X 10(-8) M. At 10 mM Mg2+ only one dissociation constant of about 1.0 X 10(-6) M is determined with both complexes. In analogy dephosphorylation of troponin P1-TI2C reduces the affinity for Mg2+ at the Ca2+/Mg2+ binding sites from 6.7 X 10(-5) M to 2.0 X 10(-3) M. Again the Mg2+-specific sites are uninfluenced. The possibility is discussed that removal of the phosphate group from troponin T allows the interaction of the N-terminal domain of troponin T with other amino acid side chains of troponin.

Alkaline Phosphatase↗