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L M Heilmeyer

Publications and source records attributed to L M Heilmeyer.

At least 37 records · Page 2Linked to original sources
PubMed1993

Identification of a new 80 k isoform of phosphatidylinositol 4-phosphate 5-kinase from bovine brain.

Phosphatidylinositol 4-phosphate 5-kinase is associated with bovine brain microsomes to an extent of approximately 65% of the total cellular enzyme activity. This membrane-associated kinase activity can be solubilized with Triton X-114. Following polyacrylamide gel electrophoresis in the presence of SDS the enzyme can be renaturated from gel slices in the presence of desoxycholate and Triton X-100. Catalytic activity appears at an apparent molecular weight of 80 k. Analysis of the reaction product formed by the 80 k protein reveals the existence of a 5-phosphotransferase, identifying the 80 k polypeptide as a new phosphatidylinositol 4-phosphate 5-kinase isoform.

Animals↗
PubMed1993

Interaction sites on phosphorylase kinase for calmodulin.

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the alpha subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the beta subunit and a peptide from the alpha subunit present in a region deleted in the alpha' isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca2+ binding to calmodulin in presence of peptides was measured. By this way, the peptides alpha 542-566, alpha 547-571, alpha 660-677 and beta 597-614 have been found to bind specifically to calmodulin. Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the alpha and beta subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in gamma as well as to two regions in alpha and beta. Exogenous calmodulin can bind to two regions in alpha and in beta. A binding stoichiometry of 0.8 mol of calmodulin/alpha beta gamma delta promoter of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23 nM calmodulin which is in the affinity range of calmodulin binding peptides from beta to calmodulin. Therefore, binding of exogenous calmodulin to beta activates the enzyme. A model for switching endogenous calmodulin between alpha, beta and gamma and modulation of ATP binding to alpha as well as Mg2+/ADP binding to beta by calmodulin is presented.

Amino Acid Sequence↗
PubMed1992

Rabbit fast skeletal muscle phospholipase C. Molecular weight determination by renaturation after polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate.

Phosphoinositide specific phospholipase C from rabbit fast skeletal muscle has been enriched ca. 1,000-fold with a specific activity of 40 mumol x min-1 x mg-1. Following SDS-PAGE, renaturation of the enzyme protein in the presence of deoxycholate allowed the determination of an apparent molecular weight of 110 kDa. Gel-filtration of the native enzyme resulted in a very similar apparent molecular weight of 115 kDa, however, associated proteins of higher molecular weight were also found. Free Ca2+ concentrations needed for half-maximal activation of PtdIns(4,5)P2, PtdIns4P and PtdIns hydrolysis are 6.3 microM, 85 microM and 1.8 mM, and the Km values for these substrates 102, 340 and 937 microM, respectively.

Animals↗
PubMed1992

Farnesylcysteine, a constituent of the alpha and beta subunits of rabbit skeletal muscle phosphorylase kinase: localization by conversion to S-ethylcysteine and by tandem mass spectrometry.

The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed.

Amino Acid Sequence↗
PubMed1992

Ordered phosphorylation of a duplicated minimal recognition motif for cAMP-dependent protein kinase present in cardiac troponin I.

Cardiac troponin I contains two adjacent serines in sequence after three arginine residues thus making up a minimally duplicated recognition motif for cAMP-dependent protein kinase. In a synthetic peptide, PVRRRSSANY, the two serine residues are phosphorylated sequentially with the intermediate formation of a monophosphorylated species according to the following reaction sequence: Peptide k1----Peptide-P k2----Peptide-P2. The calculated rat constants are: k1 = 0.435.min-1 and k2 = 0.034.min-1. Sequence analyses of the monophosphopeptide and its tryptic fragments show that the predominant monophosphoform carries phosphate at the second serine.

Amino Acid Sequence↗
PubMed1992

Compartmentalized ATP synthesis in skeletal muscle triads.

Isolated skeletal muscle triads contain a compartmentalized glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. These enzymes express activity in the structure-associated state leading to synthesis of ATP in the triadic junction upon supply of glyceraldehyde 3-phosphate or fructose 1,6-bisphosphate. ATP formation occurs transiently and appears to be kinetically compartmentalized, i.e., the synthesized ATP is not in equilibrium with the bulk ATP. The apparent rate constants of the aldolase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase reaction are significantly increased when fructose 1,6-bisphosphate instead of glyceraldehyde 3-phosphate is employed as substrate. The observations suggest that fructose 1,6-bisphosphate is especially effectively channelled into the junctional gap. The amplitude of the ATP transient is decreasing with increasing free [Ca2+] in the range of 1 nM to 30 microM. In the presence of fluoride, the ATP transient is significantly enhanced and its declining phase is substantially retarded. This observation suggests utilization of endogenously synthesized ATP in part by structure associated protein kinases and phosphatases which is confirmed by the detection of phosphorylated triadic proteins after gel electrophoresis and autoradiography. Endogenous protein kinases phosphorylate proteins of apparent Mr 450,000, 180,000, 160,000, 145,000, 135,000, 90,000, 54,000, 51,000, and 20,000, respectively. Some of these phosphorylated polypeptides are in the Mr range of known phosphoproteins involved in excitation-contraction coupling of skeletal muscle, which might give a first hint at the functional importance of the sequential glycolytic reactions compartmentalized in triads.

Adenosine Triphosphate↗
PubMed1990

Relation of phosphatidylinositol metabolism to glycolytic pathway in skeletal muscle membranes.

Skeletal muscle triads are possessing the whole set of enzymes of the phosphatidylinositol (PI)-linked signal generating pathway, PI-kinase, PI(4)P-kinase, and PI(4,5)P2-phospholipase C (PLC). The activities of these enzymes are comparable to those found in other cell types for which a functional role of the PI-pathway in intracellular signal transduction has been established. For skeletal muscle an unequivocal function and an initiating signal for Ins(1,4,5)P3-liberation is still unknown. However, the observed Ca-dependency of PLC activity suggests that here Ins(1,4,5)P3 production is a consequence rather than a cause of increasing cytosolic Ca2+. Recently, the glycolytic enzyme aldolase, whose activity can be modulated by inositol polyphosphates, has been localized in the triadic structure. The enzyme which has a high affinity to Ins(1,4)P2, Ins(1,4,5)P3 and Ins(1,3,4,5)P4, seems to be compartmentalized to the junctional foot structure from which it is released upon binding of these molecules. This phenomenon could reflect a capability for regulation of the glycolytic flux even for aldolase, especially if a non steady-state situation in the junctional gap is considered. Meanwhile we have accumulated evidence for the operation of a partial glycolytic sequence in the junctional region established by the enzymes aldolase, glyceraldehyde-3-P (GAP) dehydrogenase and phosphoglycerate kinase. This system is able to produce ATP upon oxidation of GAP and could be, because of the inositol polyphosphate-sensing abilities of aldolase, a target for the membrane associated PI-pathway. The ATP production is however transient which indicates the coupling to an ATP hydrolyzing reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

A common motif of two adjacent phosphoserines in bovine, rabbit and human cardiac troponin I.

From rabbit and human cardiac troponin I N-terminal mono and bisphosphorylated peptides were isolated which were obtained from Lys-C proteinase digests. Two adjacent phosphoserine residues could be localized in each phosphopeptide following further tryptic digestion. The previously published sequence of rabbit cardiac troponin I had to be corrected. Two adjacent phosphoserine residues are a common motif in the very similar sequences of bovine, rabbit and human cardiac troponin I. The N-terminal sequences are: AcADRSGGSTAG DTVPAPPPVR RRS(P)S(P)ANYRAY ATEPHAK (bovine), AcADESTDA-AG EARPAPAPVR RRS(P)S(P)ANYRAY ATEPHAK (rabbit), (Ac,A,D/N,G,S,S,D/N,A,A,R) EPRPAPAPIR RRS(P)S(P)-NYRAY ATEPHAK (human).

Amino Acid Sequence↗
PubMed1990

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain.

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.

Amino Acid Sequence↗
PubMed1990

Sites phosphorylated in bovine cardiac troponin T and I. Characterization by 31P-NMR spectroscopy and phosphorylation by protein kinases.

Bovine cardiac troponin isolated in a highly phosphorylated form shows four 31P-NMR signals [Beier, N., Jaquet, K., Schnackerz, K. & Heilmeyer, L.M.G. Jr (1988) Eur. J. Biochem. 176, 327-334]. Troponin I, which contains phosphate covalently linked to serine-23 and/or -24 [Swiderek, K., Jaquet, K., Meyer, H. E. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 335-342], shows three resonances. Mg2(+)-saturation of holotroponin shifts these troponin I resonances to higher fields. Direct binding of Mg2+ to the phosphate groups can be excluded. Both these serine residues of troponin I, 23 and 24, are substrates for cAMP- and cGMP-dependent protein kinases as well as for protein kinase C. Isolated bovine cardiac troponin T contains 1.5 mol phosphoserine/mol protein, indicating that minimally two serine residues are phosphorylated. One phosphoserine residue is located at the N-terminus. An additional phosphoserine is located in the C-terminal cyanogen bromide fragment, CN4, which contains covalently bound phosphate. Protein kinase C phosphorylates serine-194, thus demonstrating exposure of this residue on the surface of holotoponin.

Amino Acid Sequence↗
PubMed1990

Localization of phosphoserine residues in the alpha subunit of rabbit skeletal muscle phosphorylase kinase.

The alpha subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2+. These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A 'multiphosphorylation loop' uniquely present in this but not in the homologous beta subunit contains all the phosphoserine residues so far identified in the alpha subunit.

Amino Acid Sequence↗
PubMed1990

Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences.

A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).

Amino Acid Sequence↗
PubMed1989

Reaction of fluorescein isothiocyanate with an ATP-binding site on the phosphorylase kinase alpha subunit.

Phosphorylase kinase can be labeled specifically on the alpha subunit with fluorescein 5'-isothiocyanate (FITC) which concomitantly inactivates the enzyme (T. G. Sotiroudis and S. Nikolaropoulus (1984) FEBS Lett. 176, 421-425). Labeled peptides have been purified and their primary structure has been determined. The amino acid sequence of the fluorescein-labeled tryptic peptide is Lys-Met-Gln-Asp-Gly-Tyr-Phe-Gly-Gly-Ala-Arg. The environment of this fluorescein-labeled lysine has been determined by sequencing peptides isolated from a Staphylococcus aureus V8 digest and two further cyanogen bromide fragments of the purified [14C]carboxymethylated alpha subunit. The partial sequences obtained have then been localized in the primary structure of the alpha subunit [Zander et al. (1988) Proc. Natl Acad. Sci. USA 85, 2929-2933]. Both the incorporation of the fluorescent label and enzymatic inactivation are inhibited by ATP only at pH 7.0; ADP and AMP do not protect. Kinetic analysis reveals a competition between ATP and FITC; a Ki for ATP of 728 +/- 100 microM has been determined.

Adenosine Triphosphate↗
PubMed1988

Isolation and characterization of a highly phosphorylated troponin from bovine heart.

A modified procedure for isolation of troponin from bovine heart is described, which results in a stable and highly phosphorylated protein. 31P-NMR spectra show up to four phosphoserine signals indicating that at least four serine residues of cardiac troponin are phosphorylated in the intact organ. The hydrodynamic parameters of phosphotroponin are almost identical to those previously published. Characteristically cardiac troponin shows a strong tendency to associate that is dependent on protein concentration. Mg2+ may specifically induce an aggregation, which can be observed during sedimentation. This phenomenon seems to be analogous to the Mg2+-induced dimerization of cardiac troponin C [Jaquet, K. and Heilmeyer, L. M. G., Jr (1987) Biochem. Biophys. Res. Commun. 145, 1390-1396]. Upon Mg2+ saturation a shift of one of the four 31P-NMR signals is observed. The affinity of troponin to Ca2+ is reduced when the protein concentration is enhanced only in the presence of Mg2+. This effect of Mg2+ suggests a model for the regulation of the Ca2+-binding affinity of cardiac troponin.

Animals↗
PubMed1988

Cardiac troponin I, isolated from bovine heart, contains two adjacent phosphoserines. A first example of phosphoserine determination by derivatization to S-ethylcysteine.

Bovine cardiac troponin containing approximately 3 mol P/mol protein could be separated into its subunits without loss of phosphate. Troponin I and troponin T each contain about 1.5 mol P/mol protein. In troponin I two phosphorylated serine residues could be localized in the N-terminal region by conversion of phosphoserine to S-ethylcysteine. They are located in adjacent positions in the following sequence: -Arg-Arg-Ser(P)-Ser(P)-Ala-Asn-Tyr-Tyr-Arg-Ala-Tyr-Ala-Thr-Glu-Pro- His-Ala-Lys. This sequence shows that the first phosphoserine residue in bovine cardiac troponin I occupies a homologous position to phosphoserine-20 of rabbit cardiac troponin I.

Amino Acid Sequence↗
PubMed1988

The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.

Amino Acid Sequence↗