UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 2. Oxidation of serine in the 23-kDa NH2-terminal tryptic peptide

Myosin subfragment 1 (S1) can be specifically photomodified at the active site without polypeptide chain cleavage by irradiating the stable MgADP-orthovanadate-S1 complex with UV light above 300 nm [Grammer, J. C., Cremo, C. R., & Yount, R. G. (1988) Biochemistry (preceding paper in this issue)]. Here, the UV spectral properties of photomodified S1 were used to determine the nature and location of the photomodified residue(s) within S1. By comparison of the unusual pH dependence of the UV absorption spectrum of the photomodified S1 to that of the S1-MgADP-Vi complex as a control, the photomodified residue(s) was (were) localized to the 23-kDa NH2-terminal tryptic peptide of the heavy chain. NaBH4 reduced the photomodified S1, but not the control, to regenerate the original spectral properties and ATPase activities of the unmodified S1. Amino acid analysis of photomodified S1 reduced with NaB3H4 gave only [3H]serine, suggesting the hydroxyl group of serine had been oxidized to a "serine aldehyde". The pH dependence of the absorption spectrum of the photomodified enzyme can be explained by an equilibrium between a chromophoric enolate anion of the serine aldehyde (favored in base) and less chromophoric keto and enol forms (favored in acid). The oxidized serine(s) was (were) shown to be directly involved with the vanadate-dependent photocleavage of the S1 heavy chain previously described by Grammer et al. (1988). This serine(s) is (are) likely to be important to the binding and hydrolysis of the gamma-PO4 of ATP at the active site of S1.     

Chemical identification of serine 181 at the ATP-binding site of myosin as a residue esterified selectively by the fluorescent reagent 9-anthroylnitrile

The esterification reagent 9-anthroylnitrile (ANN) reacts with a serine residue in the NH2-terminal 23-kDa peptide segment of myosin subfragment-1 heavy chain to yield a fluorescent S1 derivative labeled by the anthroyl group (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194). The labeling was highly selective and accelerated by nucleotides. In the present study, to determine the exact location of the labeled serine residue, the labeled 23-kDa peptide fragment was isolated. The subsequent extensive proteolytic digestion of the peptide fragment yielded two labeled peptides, a pentapeptide and its precursor nonapeptide. Amino acid sequence and composition analyses of both labeled peptides revealed that the anthroyl group is attached to Ser-181 involved in the phosphate binding loop for ATP (Smith, C. A., and Rayment, I. (1996) Biochemistry 35, 5404-5417). We concluded that ANN can esterify Ser-181 selectively out of over 40 serine residues in the subfragment 1 heavy chain. Thus ANN is proved to be a valuable fluorescent tool to identify peptides containing the phosphate binding loop of S1 and to detect the conformational changes around this loop.     

Affinity labeling of the myosin ATPase with ribose-modified fluorescent nucleotides and vanadate

Ribose-modified fluorescent nucleotide analogs, 3'-O-anthraniloyl and 3'-O-(N-methylanthraniloyl) derivatives of AT(D)P, dAT(D)P, CT(D)P, UT(D)P, IT(D)P, and GT(D)P, were synthesized for use as substrates and affinity labels for the myosin ATPase [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. None of the fluorescent nucleoside triphosphate (NTP) analogs was significantly different from the corresponding natural NTP in its ability to support superprecipitation of actomyosin. When fluorescent and natural NTPs were used as substrates for the myosin subfragment-1(S-1) ATPase in the presence of 1mM vanadate ion (V1), a slight initial inhibition of the S-1 NTPase was followed by progressive inhibition to more than 60% over a period of 1 h. The apparent second-order rate constants were 0.14-0.44M-1 . s-1, suggesting the formation of the inactive fluorescent NDP-labeled S-1. After incubation of S-1 with the nucleoside diphosphate (NDP) analog in the presence of Vi, the resultant fluorescent NDP-labeled S-1 was isolated free of unbound Vi and the analog by gel filtration. The isolated complexes had stoichiometries of 0.6-1.1 NDP analog per S-1 active site. Native polyacrylamide gel electrophoresis revealed conveniently that the NDP analog is associated with S-1 as indicated by two intense fluorescent bands corresponding to S-1 isozymes. On dissociating gels, the analog was released from S-1, suggesting that the labeled S-1 is held together by strong secondary forces rather than covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of the 50-kDa peptide of myosin heads in the ATPase activity revealed by fluorescent modification with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole

The fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) reacted specifically with 1.9 lysyl residues/mol of the myosin subfragment-1 (S-1) ATPase. When 1.9 lysyl residues were modified, the K+- and Ca2+-ATPase activities were almost completely inhibited, whereas the Mg2+-ATPase activity was increased to 180% of original activity. The actin-activated Mg2+-ATPase activity was decreased to 30% of original activity by this modification. However, affinity of S-1 for actin in the presence of ATP was unchanged. The NBD fluorescence of the modified S-1 was quenched on addition of ATP, suggesting that ATP induced conformational changes around the NBD groups attached to S-1. Tryptic digestion of the modified S-1 revealed that the NBD groups are attached mainly to the 50-kDa peptide of S-1, more precisely the 45-kDa peptide. These results confirm the recent reports that the 50-kDa peptide of S-1 is involved in the myosin ATPase reaction (K?rner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G. (1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in press).     

Lysyl residues of cardiac myosin accessible to labeling with a fluorescent reagent, N-methyl-2-anilino-6-naphthalenesulfonyl chloride

Two mol of N-methyl-2-anilino-6-naphthalenesulfonyl (Mns) groups was preferentially incorporated into pig cardiac myosin in the absence of divalent metal ions, 1 mol rapidly and 1 mol slowly. In the presence of divalent metal ions. 1 mol was rapidly incorporated but subsequent incorporation was strongly suppressed. No substantial effect of incorporation of Mns groups in the presence or absence of divalent metal ions on the Ca2+- and K+-ATPase activities of myosin was found. However, the fluorescence spectra due to attached Mns groups were different in the two cases. Extensive pronase digestion of labeled myosin indicated that the Mns groups were attached predominantly to lysyl residues, regardless of the labeling conditions. Peptide mapping of the labeled myosin digested with subtilisin, pepsin or trypsin uniformly showed the selective incorporation of an Mns group into essentially one species of peptide. However, the peptide labeled in the absence of divalent metal ions was clearly different from that labeled in their presence. The present results confirm that pig cardiac myosin heavy chains contain two distinct lysyl residues, which are both accessible to labeling with Mns groups only when divalent metal ions are absent. The results also suggest that conformational changes occur around these residues when divalent metal ions are added.     

A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein

We have isolated, after limited proteolysis of the bactericidal/permeability-increasing protein (BPI) of human neutrophils, a 25-kDa fragment that possesses the bactericidal and envelope-altering activities of the 60-kDa parent protein. On a molar basis, the fragment is as potent as holo-human BPI against rough Escherichia coli, is more potent than holo-BPI against more resistant smooth E. coli, and retains the specificity of BPI toward Gram-negative bacteria. NH2-terminal amino acid sequence analysis shows that the fragment is derived from the NH2 terminus of the BPI molecule. These findings suggest that all of the molecular determinants of the antibacterial properties of BPI reside within the NH2-terminal 25-kDa segment, implying a novel structural/functional organization for a cytotoxic protein.     

Effect of peptides--structural analogs of NH2-terminal sites of fibrin alpha- and beta-chains--on specific binding of the NH2-terminal disulfide bond of fibrin with fibrinogen

Peptides Gly-Pro-Arg-Pro and Gly-His-Arg-Pro (fibrin alpha- and beta-chain NH2-terminal analogs, respectively) are studied for their effect on fibrinogen (F) and fibrin NH2-terminal disulphide knot (N-DSK) specific binding. Both peptides are found to inhibit the formation of soluble and insoluble F-N-DSK-complexes through inhibition of the interdomain D-E-binding. Gly-Pro-Arg-Pro is much more potent inhibitor than Gly-His-Arg-Pro. Lowering the insoluble F-N-DSK-copolymer quantity by concentration-dependent way these peptides do not change its composition described by the formula [F(N-DSK)2]n. Invariability of fibrinogen and N-DSK copolymer structure is asserted. In this structure neighbouring fibrinogen molecules are bound by two N-DSK molecules via the D1-E1 and D2-E2 binding sites.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Affinity labeling of a subsite of Taka-amylase A by the fluorescent reagent o-phthalaldehyde

A cross-linked modification of Lys residue located at the subsite of the enzyme active site of Taka-amylase A was attained by the use of the fluorescent reagent of o-phthalaldehyde (OPA). The fluorescence and uv absorption at 337 nm derived from the isoindole ring, which was produced by cross-linking through the epsilon-amino group of Lys and the thiol group of the Cys residue, provided the evidence for the OPA-mediated inactivation of Taka-amylase A. Kinetic analysis showed that 1 mol of OPA per mole of enzyme was incorporated, which corresponded closely with the value obtained by the uv absorption. Because the OPA inactivation was retarded by the substrate analog of alpha-cyclodextrin, OPA modification was classified as a type of affinity labeling reaction. A remarkable increase in the pI value from 4.0 to 5.6 upon the modification led to clear separation of the modified enzyme from the native Taka-amylase A by a DEAE-Sephacel column and led to the charge isomer pattern on gel electrophoresis performed according to the method of Hedrick and Smith. Moreover, the affinity gel electrophoresis showed that the modified enzyme completely lost the affinity for the substrate soluble starch, which indicated that the subsite modification occurred.     

Factor XIII-mediated cross-linking of NH2-terminal peptide of alpha 2-plasmin inhibitor to fibrin

The NH2-terminal 12-residue peptide of alpha 2-plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Gly-Leu-Lys-NH2 . AcOH, was found to be a good substrate for plasma transglutaminase (activated blood coagulation factor XIII) and rapidly incorporated into fibrin by the enzyme. A high concentration of the peptide inhibited the enzyme-mediated cross-linking of alpha 2-plasmin inhibitor to fibrin probably by competing with the inhibitor for the same site of fibrin alpha-chain.     

Studies on the structure of rabbit muscle aldolase: Ordering of the tryptic peptides; Sequence of 164 amino acid residues in the NH2-terminal BrCN peptide

The sequence of 164 amino acid residues in the NH₂-terminal BrCN peptide of rabbit muscle aldolase has been determined. The information has permitted location of the following amino acid residues involved in the catalytic activity or in maintaining the structural integrity of the enzyme: Cys-72, forms a disulfide bridge with Cys-336 in the COOH-terminal segment on inactivation of the enzyme by oxidation; Lys-107, forms a Schiff base with pyridoxal phosphate upon inactivation of aldolase by this reagent; Cys-134 and Cys-177, buried, do not react with SH-reagents in the native enzyme.     

Characterization of Vibrio cholerae neuraminidase by a novel mechanism-based fluorescent labeling reagent

Vibrio cholerae neuraminidase (VCNA) plays a significant role in the pathogenesis of cholera by removing sialic acid residues from higher-order gangliosides to an unmasked GM1, the essential receptor for cholera toxin. Here we report that a novel mechanism-based fluorescent labeling reagent, 5-acetamido-2-(4-N-5-dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-3,5-dideoxy-d-glycero-alpha-d-galacto-2-nonulopyranosonic acid (1), becomes a unique irreversible inhibitor of VCNA. Characterization of an inactivated VCNA by MALDI-TOF/TOFMS analysis revealed that the Asp-576 and Arg-577 residues, which are located within the (576)DRFF(579) sequence, were specifically labeled with this suicide-type fluorescent substrate. Neither Asp-576 nor Arg-577 has ever been known to contribute to a specific residue in the rigid and highly conserved active site of VCNA investigated by crystallographic analysis, suggesting that a flexible beta-turn structure containing this sequence may have a crucial role in the dynamic nature of substrate recognition and catalytic action by VCNA.     

Myristic acid is the NH2-terminal blocking group of the 43-kDa protein of Torpedo nicotinic post-synaptic membranes

The NH2-terminal blocking group of the 43-kDa peripheral membrane protein (43-kDa protein) of Torpedo post-synaptic membranes has been identified as myristic acid. To identify that blocking group pure 43-kDa protein was digested with trypsin and the blocked tryptic peptide was isolated by reverse phase HPLC. That peptide coeluted with and had the same amino acid composition as a synthetic peptide, myristoyl-Gly-Gln-Asp-Gln-Thr-Lys, the structure of the amino terminus predicted from the protein sequence deduced from a cDNA clone. The presence of myristate was confirmed by the precise molecular mass of the peptide, 886.5266, determined by fast atom bombardment mass spectroscopy.     

Plasma desorption mapping of the tryptic digest of 23-kDa recombinant human growth hormone

Plasma desorption mass spectrometry has been used to map the tryptic fragments from the 23-kDa recombinant human growth hormone protein. The unfractionated tryptic digest was adsorbed directly onto a nitrocellulose sample foil and mass spectra were obtained in both the positive and the negative ion mode. The adsorbed sample was then washed with deionized water and its mass spectrum was again obtained. The latter spectrum revealed tryptic fragments that were not observed in the spectra of the unwashed sample, which can be attributed (to some extent) to the removal of hydrophilic residues during washing. From this study a protocol, aimed at the complete mapping of tryptic fragments, is outlined.     

Selective fluorescent labeling of the 50-, 26-, and 20-kilodalton heavy chain segments of myosin ATPase

7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin (CPI), rhodamine B isothiocyanate (RITC), and 4-bromomethyl-6,7-dimethoxycoumarin (BDMC), fluorescent reagents that can react covalently with amino or sulfhydryl groups, have been used to label myosin subfragment-1 (S-1) ATPase. The conditions under which CPI, RITC, and BDMC selectively label the 50-, 26-, and 20-kDa segments of the S-1 heavy chain, respectively, are described. CPI and RITC labeling little affects the ATPase activities of S-1 in the presence and absence of actin. BDMC labeling activates the Ca2+- and Mg2+-ATPases of S-1, and abolishes the K+-EDTA-ATPase. The three S-1 derivatives fluoresce strongly even under acidic conditions, suggesting the wide applicability of these fluorescent reagents as selective labels for the three segments of the S-1 heavy chain.     

The interaction of anions with hemoglobin carbamylated on specific NH2-terminal residues.

Carbamylation of the NH2-terminal residues of the beta chains on hemoglobin (alpha2beta2c) leads to a reduced but still significant binding of 2,3-diphosphoglycerate, but has no effect on the oxygen-linked binding of chloride or phosphate, both of which are thought to bind to some of the same residues as the organic phosphate. Studies by others have shown that the binding of inorganic anions is not diminished in either horse hemoglobin or in hemoglobin Little Rock, in which four of the six other binding sites (histidine residues) for organic phosphates are replaced by glutamine residues. We suggest, therefore, that lysines 82 of the beta chains, which are the remaining 2 residues in the binding crevice for the organic phosphate, and which are invariant in the known sequences of mammalian hemoglobins, may be the primary binding site for inorganic anions. The extent of inhibition of gelation by increasing ionic strength is identical for the hybrids alpha2beta2, alpha2cbeta2, and alpha2beta2c of hemoglobin S. These results indicate the NH2-terminal residues of the chains are not involved in primary electrostatic interactions during aggregation of deoxyhemoglobin S.     

Inhibitory effect of ATP analogs and actin on the modification of myosin subfragment 1 with 9-anthroylnitrile

The fluorescent probe, 9-anthroylnitrile (ANN), can selectively attach to Ser-180 at the ATP-binding site of subfragment 1 (S1) of skeletal muscle myosin [J. Biol. Chem. 278 (2003) 31891]. We have found that MgATP, MgATPgammaS, MgADP.AlF(4) or MgPP(i), but not MgADP, inhibit the incorporation of ANN into S1. The inhibitory effect of the nucleotide gamma-phosphate group (or its analog) on the modification of S1 with ANN can be explained by the contribution of Ser-180 to the binding of the nucleotide gamma-phosphate at the active site of S1. We have also observed that the incorporation of ANN into S1.MgADP complex is inhibited by actin. These experimental data strongly support the existence of nucleotide-promoted conformational changes revealed by crystal structures of S1 complexes with various nucleotide analogs. They also convincingly show an effect of actin on the environment of Ser-180 at the nucleotide binding site of S1.     

Interactions between G-actin and myosin subfragment 1: immunochemical probing of the NH2-terminal segment on actin

The role of the N-terminal segment of actin in myosin-induced polymerization of G-actin was studied by using peptide antibodies directed against the first seven N-terminal residues of alpha-skeletal actin. Light scattering, fluorescence, and analytical ultracentrifugation experiments showed that the Fab fragments of these antibodies inhibited the polymerization of G-actin by myosin subfragment 1 (S-1) by inhibiting the binding of these proteins to each other. Fluorescence measurements using actin labeled with pyrenyliodoacetamide revealed that Fab inhibited the initial step in the binding of S-1 to G-actin. It is deduced from these results and from other literature data that the initial contact between G-actin and S-1 involves residues 1-7 on actin and residues 633-642 on the S-1 heavy chain. This interaction appears to be of major importance for the binding of S-1 and G-actin. The presence of additional myosin contact sites on G-actin was indicated by concentration-dependent recovery of S-1 binding to G-actin without displacement of Fab. The reduced Fab inhibition of S-1 binding to polymerizing and polymerized actin is consistent with the tightening of acto-S-1 binding at these sites or the creation of new sites upon formation of F-actin.     

A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors

The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.