Ludwig M. G. Heilmeyer

Sequence analysis of phosphoserine-containing peptides: Modification for picomolar sensitivity

Sequencing of phosphoserine-containing peptides yields normally no identifiable PTH-derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one-step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as β-methylaminoalanine (MAA), S-ethanolcysteine or S-ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas-phase sequencer equipped with an on-line PTH amino acid analyzer.Sequencing of phosphoserine‐containing peptides yields normally no identifiable PTH‐derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one‐step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as β‐methylaminoalanine (MAA), S‐ethanolcysteine or S‐ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas‐phase sequencer equipped with an on‐line PTH amino acid analyzer.

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle

In this study we demonstrate the existence of a protein with properties of the voltage‐dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross‐react with four different VDAC monoclonal antibodies. (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform. (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [14C]DCCD. (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR. (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage‐dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD. We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP. Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger. These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport. Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.

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 RR ANYRAY ATEPHAK (bovine), AcADESTDA‐AG EARPAPAPVR RR ANYRAY ATEPHAK (rabbit), (Ac,A,D/N,G,S,S,D/N,A,A,R) EPRPAPAPIR RR ‐NYRAY ATEPHAK (human).

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 k 1|→ Peptide‐P k 2|→ Peptide‐P2. The calculated rate constants are: k 1 = 0.435 min−1 and k 2 = 0.034 min−1. Sequence analyses of the monophosphopeptide and its tryptic fragments show that the predominant monophosphoform carries phosphate at the second serine.

Differentiation of two catalytic sites on phosphorylase kinase for phosphorylase b and troponin T phosphorylation.

Troponin which regulates the actomyosin ATPase (reviewed [l] ) has been shown to exist in phosphorylated and non-phosphorylated forms. The subunit, T, which mediates the binding of the holotroponin complex to tropomyosin can be preferentially phosphorylated by the Ca’+-dependent phosphorylase kinase (EC 2.7.1.38) [24] and only to a very small extent by the CAMP-dependent kinase [2,5]. Phosphorylase kinase, a large oligomer of the composition o&y4 [6,7] or a&/_&y4 [8,9] mol. wt 1.25 X 106), phosphorylates the troponin subunit, T, ca. 300 times slower than phosphorylase b [3] . This could be due to a lower substrate specificity of phosphorylase kinase for the T subunit or to a contamination of this enzyme with another protein kinase as proposed [lo] . Alternatively, phosphorylase kinase may be a double or multiheaded enzyme which is composed of several kinases. It will be shown here that phosphorylase kinase and troponin T kinase activity are enriched together. By eluting the enzyme from a DEAE column the activity ratio is not constant which might be correlated with a small change in the ratio of the subunits (o t a’) : 0 : 7. Antibodies against phosphorylase kinase inhibit both, the troponin and phosphorylase kinase activity. However, the inhibition pattern differs with both substrates. In addition troponin does not inhibit the conversion of phosphorylase b to (1. It is concluded that troponin and phosphorylase are phosphorylated by two different catalytic centers. These may be situated on the same holoenzyme.

The identification of the phosphorylated 150/160-kDa proteins of sarcoplasmic reticulum, their kinase and their association with the ryanodine receptor

In the present work we studied the relationship between the phosphorylated 150- and 160-kDa proteins and other SR proteins in the 150,000-170,000 range of molecular masses. on SDS-PAGE, the identification of their kinase, as well as the purification and structural interactions between these proteins and the rynodine receptor (RyR). The phosphorylated 150-kDa protein was identified as sarcalumenin based on: (a) its cross-reactivity with three different monoclonal antibodies specific for sarcalumenin. (b) its mobility in SDS-PAGE which was modified upon digestion with endoglycosidase H, (c) its elution from lentil-lectin column by alpha-methyl mannoside, (d) its resistance to trypsin, (e) its ability to bind Ca2+ and to stain blue with Stains-All. The phosphorylated 160-kDa protein was identified as the histidine-rich Ca2+ binding protein (HCP) based on: (a) its Ca(2+)-binding property and staining blue with Stains-All, (b) phosphorylation with the catalytic subunit of cAMP-dependent kinase. (c) its increased mobility in SDS-PAGE in the presence of Ca2+ (d) its heat stability and (e) its partial amino acid sequence. The endogenous kinase was identified as casein kinase II (CK II) based on the inhibition of the endogenous phosphorylation 160/150-kDa proteins by heparin, 5.6-dichlorobenzimidazole riboside, polyaspartyl peptide and hemin, and its ability to use [gamma-32P]GTP as the phosphate donor. The association of CK II with SR membranes, was demonstrated using specific polyclonal anti-CK II antibodies. The luminal location of CK II is suggested because CK II was extracted from the SR by l M NaCl only after their treatment with hypotonic medium, and CK II activity was inhibited with the charged inhibitors heparin and polyaspartyl peptide only after their incubation with the SR in the presence of NP-40. The 160- and 150-kDa proteins were purified on spermine-agarose column, and were phosphorylated by CK II. Like the endogenous phosphorylation of the 150/160-kDa proteins in SR. the phosphorylation of the purified proteins by CK II was inhibited by La3+ (Cl50 = 4 microM) and hemin. The results suggest the phosphorylation of the luminally located sarcalumenin and HCP with CK II.

Bisphosphorylation of cardiac troponin I modulates the Ca2+-dependent binding of myosin subfragment S1 to reconstituted thin filaments

We have reconstituted thin filaments comprising pyrene‐labelled actin (pyr‐actin), tropomyosin (Tm) and cardiac troponin (cTn). cTn was isolated in two defined phosphorylation states; completely dephosphorylated on all subunits and with only the cTnI subunit bisphosphorylated. The thin filament was saturated with cTn at a pyr‐actin/Tm/cTn ratio of 7:1:1. The calcium‐dependent binding of S1 to thin filaments was measured in a stopped‐flow spectrophotometer and the dependence of the observed rate constant on [Ca2+] fitted to the Hill equation. The only significant difference between the two phosphorylation states of the filaments was a 0.36 decrease in the pCa50 on bisphosphorylation.

Endogenous, Ca2+-dependent cysteine-protease cleaves specifically the ryanodine receptor/Ca2+ release channel in skeletal muscle

The association of an endogenous, Ca2+-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and β-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.1 μm and maximal activity at 10 μm. Mg2+ is also an activator of this enzyme (CI50=22 μm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type.This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca2+-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. A possible involvement of this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity is discussed.

Microanalysis and distribution of cardiac troponin I phospho species in heart areas.

Sequential phosphorylation and dephosphorylation of cTnI by the cAMP dependent protein kinase and by protein phosphatase 2A, respectively, produce the non-, mono- and bisphosphorylated species (Jaquet et al., 1995, Eur. J. Biochem. 231, 486-490). The aim of this study was to determine these forms even in small tissue samples, e.g. in biopsy probes of approximately 30 mg which would allow to define the phosphorylation state of cTnI in heart areas. In order to do so a micro isolation procedure for cTnI had to be established. cTnI is extracted from small bovine, rabbit and human heart tissue samples (30-100 mg) under special conditions avoiding dephosphorylation and is isolated by affinity chromatography on cTnC Sepharose. All three species, the bis-, mono- and dephospho cTnI, are precipitated quantitatively by acetone, then they are separated by non-equilibrium isoelectric focusing and quantified by scanning densitometry. The method presented here allows to quantify the three cTnI species reproducibly. No other phosphorylated species are detected. Truncated cTnI forms of each phospho species are found in human biopsy samples due to removal of a approximately 36 amino acid peptide from the C-terminus. In bovine, human and rabbit heart the pattern of the three cTnI phospho species is characteristic for left and right atrium, left and right ventricle and septum.

Phosphofructokinase is a calmodulin binding protein

A trial to purify myosin light chain kinase from crude myosin led to the isolation of a M r 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.