Sulfation of a gamma-chain variant of human fibrinogen

Biosynthetic sulfation of human fibrinogen was investigated using a hepatoma-derived cell line in culture. Very little [35]sulfate was incorporated into the major forms of the A alpha, B beta, or gamma-chains of fibrinogen, but there was a labeled peptide chain with electrophoretic mobility intermediate between the B beta and gamma-chains. Base hydrolysis of the sulfate-labeled product released tyrosine sulfate. The labeled peptide is identified as a form of gamma-chain by its resistance to proteolysis during extended periods of incubation, in contrast with A alpha and B beta-chains which are converted to smaller forms. The results indicate that human fibrinogen contains tyrosine sulfate primarily within a variant form of the gamma-chain.     

Factors affecting gamma-chain multimer formation in cross-linked fibrin.

The major covalently linked multimolecular D fragments found in plasmic digests of factor XIIIa cross-linked fibrin formed under physiological pH and ionic strength conditions consist of D dimers, D trimers, and D tetramers. These fragments are linked by epsilon-amino-gamma-glutamyllysine bonds in the carboxy-terminal regions of their gamma chains, which had originated in the cross-linked fibrin as gamma dimers, gamma trimers, and gamma tetramers, respectively. In this study, factors affecting the degree and rate of formation of these three classes of cross-linked gamma chains were determined by analyzing the D-fragment content of plasmic digests of cross-linked fibrin that had been sampled after all gamma-chain monomers had been consumed in the cross-linking process. D trimers and D tetramers, expressed as a proportion of the total D-fragment content, both increased at the expense of the D-dimer population as a function of increasing factor XIII concentration, the time of cross-linking, or the CaCl2 concentration. Their levels decreased as the ionic strength was raised by NaCl addition. However, the ionic strength effect could be reversed by concomitantly raising the CaCl2 concentration. Digests of clots prepared from recalcified fresh citrated plasma also contained each type of cross-linked D fragment, and the proportion of D trimers and D tetramers in the digest increased with increasing clot incubation time. These results indicate that gamma-trimer and gamma-tetramer formation is a dynamic physiological process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implications of the invariant gamma-chain on the intracellular transport of class II histocompatibility antigens

We have examined the role of the invariant gamma-chain on the intracellular transport of human class II histocompatibility antigens. mRNA was selected by hybridization to cDNA corresponding to class II alpha-, beta-, and gamma-chains, and the obtained mRNA fractions were injected individually and in various combinations into X. laevis oocytes. Translation products were isolated after various periods of chase, and their carbohydrate moieties were analyzed to monitor the subcellular localization of polypeptide chains. A mixture of alpha-, beta-, and gamma-chains, or gamma-chains alone, were transported and glycosylated to the same extent as in a B lymphoblastoid cell line. However, although alpha- and beta-chains formed a complex in the absence of the gamma-chain, the transport of this complex was slowed down. Furthermore, the glycosylation of alpha- and beta-chains appeared incomplete. Thus, the invariant gamma-chain seems to play a crucial role for the rate of transport and glycosylation of class II alpha- and beta-chains.     

Localization of segments essential for polymerization and for calcium binding in the gamma-chain of human fibrinogen

We have isolated an intermediate plasmic degradation product, D2, of fibrinogen that does not inhibit the polymerization of fibrin monomer but does bind Ca2+. Fibrinogen was digested to a limited extent with plasmin in the presence of Ca2+, and a "large" fragment D (fragment D1A) was isolated with a gamma-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its gamma-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the gamma-chain. Fragment D2 (with its gamma-chain containing residues 86-335/356) was isolated from an intermediate digest in the presence of EGTA. The Lys-338-Cys-339 peptide bond of the gamma-chain is intact in this preparation of D2, even though it is split in the isolated peptide gamma303-355 (with an intact disulfide bond at Cys-326-Cys-339). Fragment D2 does not interfere with the polymerization of fibrin monomer, whereas fragment D1 is a potent inhibitor of this polymerization. We conclude that the gamma-chain segment 356/357-411, present in fragment D1 but absent from fragment D2, is essential for maintenance of a polymerization site located in the outer (D) nodule of fibrinogen. This segment (356/357-411) is longer than two shorter ones reported earlier [Olexa, S.A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B.H., Váradi, A., & Scheraga, H.A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5980-5984]; the data for the earlier reports are reinterpreted here. Finally, fragment D2 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its gamma-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment gamma 303-355/356 plays a crucial role in Ca2+ binding.     

Amino acid sequence of the carboxy-terminal cyanogen bromide peptide of the human fibrinogen beta-chain: homology with the corresponding gamma-chain peptide and presence in fragment D.

The carboxy-terminal cyanogen bromide fragment of the human fibrinogen beta-chain has been isolated and its structure determined. It is a nonapeptide with the sequence Lys-Ile-Arg-Pro-Phe-Phe-Pro-Gln-Gln and is homologous with a portion of the carboxy-terminal cyanogen bromide fragment of the gamma-chain. The peptide has also been isolated in full yield from cyanogen bromide digests of the plasmin-derived fragment D, indicating that the carboxy-terminal region of the beta-chain is resistant to plasmin digestion. In contrast, a small portion of the corresponding gamma-chain carboxy-terminal region was missing in the same fragment D.     

Cross-linked amino acids in the protein pair S13-S19 and sequence analysis of protein S13 of Bacillus stearothermophilus ribosomes

The 30S ribosomal subunits from Bacillus stearothermophilus were cross-linked under native conditions with the bifunctional reagent diepoxybutane. The dominant protein-protein cross-link in the 30S ribosomal subunit between proteins S13 and S19 [Brockm?ller, J., & Kamp, R.M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935] was isolated on a preparative scale. The presence of a single cross-link site between cysteine-83 of protein S13 and histidine-68 of protein S19 was established by microsequence analysis of isolated cross-linked peptides. This cross-link site was further confirmed by different analytical methods including fast atom bombardment mass spectrometry of the cross-linked peptide. The cross-linking site is located in the highly conserved C-terminal regions of proteins S13 and S19. In addition, the complete amino acid sequence of protein S13 from B. stearothermophilus is determined. Sequence comparison with the homologous Escherichia coli protein S13 revealed 58% identical amino acid residues.     

Role of associated gamma-chain in tyrosine kinase activation via murine Fc gamma RIII.

Type III receptors for the Fc portion of IgG (Fc gamma RIII), initially characterized on macrophages and NK cells, are also expressed on several pre-B cell lines. Surface expression of Fc gamma RIII requires the association of the ligand binding alpha-chain with homodimeric gamma-chains. Type II Fc gamma R is homologous to Fc gamma RIII alpha-chain in the extracellular portion and differs in the transmembrane and cytoplasmic domains. The role of Fc gamma R in cell activation was investigated by expressing Fc gamma RIII and the lymphocyte-specific b1 isoform of Fc gamma RII (Fc gamma RIIb1) in an Fc gamma R-negative, sIgG-positive B-cell line. We found that, in contrast to Fc gamma RIIb1, Fc gamma RIII triggers the same events of cell activation as sIG i.e. Ca2+ mobilization, tyrosine phosphorylation and IL-2 secretion. By expressing cytoplasmic domain-lacking Fc gamma RIII alpha-chain in the absence or in the presence of gamma-chains, we demonstrated that cell activation via Fc gamma RIII requires the co-expression of gamma-chains, and is independent of the cytoplasmic portion of the alpha-chain. Furthermore, the cytoplasmic portion of the gamma-chain, fused to the extracellular and transmembrane domains of Fc gamma RII confers on the chimeric receptor the ability to trigger cell activation. Mutation of one tyrosine residue in the cytoplasmic domain of the gamma-chain prevented triggering of cytoplasmic signals. We therefore demonstrate that a tyrosine-containing motif, present in the cytoplasmic domain of the associated gamma-chain, is necessary and sufficient to trigger cell activation via Fc gamma RIII.     

Identification of the site of interaction between cytochrome c3 and ferredoxin using peptide mapping of the cross-linked complex.

Structural studies carried out on a cross-linked complex between cytochrome c3 and ferredoxin I, both isolated from Desulfovibrio desulfuricans Norway, allowed the identification of the site of interaction between the two redox proteins. Staphylococcus aureus proteinase and chymotrypsin digestions led to characterization of peptides containing both cytochrome c3 and ferredoxin sequences. The cytochrome c3 sequences involved in the three isolated cross-linked peptides contained several lysine residues localized around the heme 4 crevice. This analysis stressed the peculiar role of lysines 100, 101, 103, 104 and 113, which could be considered as major cross-link sites, as opposed to the lysines 75, 79 and 82, which could be considered as minor cross-link sites. One cross-linked peptide, containing two ferredoxin sequences joined to one cytochrome c3 sequence, had been isolated, suggesting the possibility of more than one cross-link per covalent complex. All these results led to the identification of heme 4 of cytochrome c3 as the site of interaction for the ferredoxin I. This study confirms the proposal that could be deduced from the hypothetical structure of the complex built by computer graphics modelling (Cambillau, C., Frey, M., Mosse, J., Guerlesquin, F. and Bruschi, M. (1988) Proteins: struct., funct. genet. 4, 63-70).     

Synthesis of a cyclic fibrin-like peptide and its analysis by fast atom bombardment mass spectrometry

For immunochemical purposes, a cyclic 12-peptide was synthesized to model the gamma-gamma-chain cross-link site in human fibrin. The model was based upon the structure proposed by Chen & Doolittle (Biochemistry (1971) 10, 4486-4491) which is characterized by two reciprocating epsilon-(gamma-Glu)Lys bonds between adjacent fibrin gamma-chains oriented in an antiparallel manner. To achieve the antiparallel orientation of the peptide backbone, Pro and Gly were inserted at positions 6 and 7 of the linear 12-peptide: acetyl-Gly-Glu-Gln-His-His-Pro-Gly-Gly-Gly-Ala-Lys-Gly-amide. The insertions were made to facilitate a reverse turn of the peptide during the last synthetic step, which was formation of the epsilon-(gamma-Glu)Lys bond between Glu at position 2 and Lys at position 11 with diphenylphosphorylazide. The resulting cyclic peptide represented half of the symmetrical cross-linked region in clotted fibrin. Following purification by HPLC, both linear and cyclic 12-peptides were analyzed by fast atom bombardment mass spectrometry. Abundant molecular protonated ions were observed for both peptides. In addition, the amino acid sequence of the linear peptide and the location of the epsilon-(gamma-Glu)Lys bond in the cyclized peptide could be verified.     

Fibrinogen Nagoya, a replacement of glutamine-329 by arginine in the gamma-chain that impairs the polymerization of fibrin monomer

Structural studies on a hereditary abnormal fibrinogen, fibrinogen Nagoya (Takamatsu, J., Ogata, K., Kamiya, T., Koie, K., Takagi, T., & Iwanaga, S. (1979) Thromb. Haemost. 42, 78), were performed to identify the abnormality responsible for the impaired polymerization of fibrin monomer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, fibrinogen Nagoya showed the presence of an extra protein band with an apparent molecular weight of 49,500 in addition to the normal three subunit chains. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of one of the CNBr fragments derived from fibrinogen Nagoya indicated that Gln-329 in the gamma-chain had been replaced by Arg. This substitution can be explained by a single nucleotide change in the codon for Gln-329 (CAG----CGG). We conclude that Gln-329 in the gamma-chain is indispensable for the normal polymerization of fibrin monomer.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.     

1H NMR sequential assignments and secondary structure analysis of human fibrinogen gamma-chain C-terminal residues 385-411

The human fibrinogen gamma-chain, C-terminal fragment, residues 385-411, i.e., KIIPFNRLTIGEGQQHHLGGAKQAGDV, contains two biologically important functional domains: (1) fibrinogen gamma-chain polymerization center and (2) platelet receptor recognition domain. This peptide was isolated from cyanogen bromide degraded human fibrinogen and was investigated by 1H NMR (500 MHz) spectroscopy. Sequence-specific assignments of NMR resonances were obtained for backbone and side-chain protons via analysis of 2D NMR COSY, double quantum filtered COSY, HOHAHA, and NOESY spectra. The N-terminal segment from residues 385-403 seems to adopt a relatively fixed solution conformation. Strong sequential alpha CH-NH NOESY connectivities and a continuous run of NH-NH NOESY connectivities and several long-lived backbone NH protons strongly suggest the presence of multiple-turn or helix-like structure for residues 390 to about 402. The conformation of residues 403-411 seems to be much less constrained as evidenced by the presence of weaker and sequential alpha CH-NH NOEs, the absence of sequential NH-NH NOEs, and the lack of longer lived amides. Chemical shifts of resonances from backbone and side-chain protons of the C-terminal dodecapeptide, residues 400-411, differ significantly from those of the parent chain, suggesting that some preferred C-terminal conformation does exist.     

Identification of sites of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen cross-linking in Escherichia coli 23S ribosomal ribonucleic acid

The reagent 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) was used to cross-link 23S rRNA from Escherichia coli under 50S ribosomal subunit reconstitution conditions. Following partial digestion of the RNA with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate fragments derived from the cross-linked sites. These fragments were analyzed by digestion with ribonucleases T1 and A and their positions in the 23S RNA sequence identified. Fragment a1 (positions 1325-1426) is cross-linked to a2 (positions 1574-1623); fragment b1 (positions 1700-1731) is cross-linked to b2 (positions 1732-1753); and a cross-link is formed within fragment c (or c') (positions 863-916). In the latter case, the cross-link was located precisely, linking residues C867 and U913. All three HMT-mediated cross-links are consistent with a proposed secondary structure model for 23S RNA [Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R., Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A., Gupta, R., & Woese, C. R. (1981) Nucleic Acids Res. 9, 6167-6189].     

Altered murein composition in a DD-carboxypeptidase mutant of Streptococcus pneumoniae.

The muropeptide composition of a Streptococcus pneumoniae mutant in which the DD-carboxypeptidase (penicillin-binding protein 3) gene was interrupted by plasmid insertion close to the 3' end of the gene was examined. Extensive compositional changes were observed: the linear pentapeptide, a minor component of the parental cells, became the most abundant monomeric peptide in the mutant wall, while the proportion of tripeptides that represent the main monomers in the parental cells was greatly reduced. The amount of the major dimer of parental cells, the directly cross-linked tri-tetrapeptide, was also reduced by a factor of 4. It was partially replaced by a novel dimer: the cross-linked product of a linear pentapeptide and a pentapeptide carrying a serylalanine dipeptide substituent on the epsilon-NH2 group of its lysine residue. This dimer together with two other dimeric peptides, each containing the serylalanine cross bridge, became the quantitatively major components of the mutant peptidoglycan.     

The complete amino acid sequence of a post-synaptic neurotoxin isolated from the venom of the Australian death adder snake Acanthophis antarcticus

1. A lethal neurotoxin (acanthophin d) was isolated from the venom of the Australian death adder snake Acanthophis antarcticus. 2. Acanthophin d consisted of a single polypeptide chain of 74 amino acid residues cross-linked by five disulphide bridges. 3. The results of neurophysiological experiments on murine phrenic nerve hemi-diaphragm preparations were consistent with irreversible post-synaptic blockage of neuromuscular transmission by acanthophin d.     

Induction of helical structure in a heptapeptide with a metal cross-link: modification of the Lifson-Roig helix-coil theory to account for covalent cross-links

A short peptide, acetyl-AHAAAHA-carboxamide, has been synthesized and the histidines cross-linked with a cis-tetraammineruthenium(III) moiety. In the absence of the Ru(III) cross-link, the heptapeptide is essentially structureless, as judged by circular dichroism, NMR chemical shift, and NMR-monitored hydrogen deuterium exchange data. The presence of the cis-Ru(III) cross-link is confirmed by mass spectral data and the characteristic pH dependence of the UV-vis spectrum of the cis-(bis-(imidazole))ruthenium(III) unit. Circular dichroism data indicate that the Ru(III) cross-linked heptapeptide is approximately 37% helical at 0 degrees C. The NMR spectrum of the cross-linked peptide has been fully assigned using TOCSY and ROESY experiments. ROE interactions and J-coupling data provide evidence for helical structure. NMR-monitored hydrogen-deuterium exchange data for the Ru(III)-cross-linked peptide, resolved at the level of the individual amides, give larger protection factors at the ends than in the center of the helix. Steric and polarization effects of the Ru(III) cross-link are proposed to cause this unusual apparent protection pattern. A modification to the Lifson-Roig helix-coil model to account for the effect of the i,i+4 Ru(III) cross-link on the helix-coil transition of a peptide is presented. The model provides an excellent fit to the temperature dependence of the circular dichroism spectrum of the Ru(III)-cross-linked peptide. The modified model indicates that the effect of the cross-link on the nucleation parameter, v(2), is modest (about 7-fold) for residues bounded by the cross-link. Significant increases in the propagation parameter, w, occur for residues within the cross-link. The modification to the Lifson-Roig model accounts for the effect of a Ru(III) cross-link on the circular dichroism spectrum of a previously reported 17 residue peptide.     

The crystal structure of fragment double-D from cross-linked lamprey fibrin reveals isopeptide linkages across an unexpected D-D interface

The crystal structure of fragment double-D from factor XIII-cross-linked lamprey fibrin has been determined at 2.9 A resolution. The 180 kDa covalent dimer was cocrystallized with the peptide Gly-His-Arg-Pro-amide, which in many fibrinogens, but not that of lamprey, corresponds to the B-knob exposed by thrombin. The structure was determined by molecular replacement, a recently determined structure of lamprey fragment D being used as a search model. GHRPam was found in both the gamma- and beta-chain holes. Unlike the situation with fragment D, the crystal packing of the cross-linked double-D structure exhibits two different D-D interfaces, each gamma-chain facing gamma-chains on two other molecules. One of these (interface I) involves the asymmetric interface observed in all other D fragments and related structures. The other (interface II) encompasses a completely different set of residues. The two abutments differ in that interface I results in an "in line" arrangement of abutting molecules and the interface II in a "zigzag" arrangement. So far as can be determined (the electron density could only be traced on one side of the cross-links), it is the gamma-chains of the newly observed zigzag units (interface II) that are joined by the reciprocal epsilon-amino-gamma-glutamyl cross-links. Auspiciously, the same novel D-D interface was observed in two lower-resolution crystal structures of human double-D preparations that had been crystallized under unusual circumstances. These observations show that double-D structures are linked in a way that is sufficiently flexible to accommodate different D-D interfaces under different circumstances.     

Packing of the transmembrane helices of Na,K-ATPase: direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking

Spatial relationships among the transmembrane (TM) segments of alpha- and beta-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic alpha-subunit contains 10 TM alpha-helices (H1-H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of beta-subunit (Hbeta). Previously, the cross-linking products containing the beta-subunit and two fragments of alpha-subunit (the N-terminal containing H1-H2 helices and the C-terminal containing H7-H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528-26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855-7858]. Here, we have shown that Cu(2+)-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of beta-subunit and a 22-kDa C-terminal alpha-fragment containing H7-H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853-Met942 alpha-fragment and Ala5-Met56 beta-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hbeta and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hbeta and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of alpha-fragments Lys947-Met963 and Tyr974-Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of alpha-subunit.