The intramolecular autoglucosylation of monomeric glycogenin

The ability of monomeric glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant glycogenin. We determined that monomer glycogenin exists in solution at concentration below 0.60-0.85 μM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step.     

Evidence for glycogenin autoglucosylation cessation by inaccessibility of the acquired maltosaccharide

Glycogenin initiates the biosynthesis of proteoglycogen, the mammalian glycogenin-bound glycogen, by intramolecular autoglucosylation. The incubation of glycogenin with UDP-glucose results in formation of a tyrosine-bound maltosaccharide, reaching maximum polymerization degree of 13 glucose units at cessation of the reaction. No exhaustion of the substrate donor occurred at the autoglucosylation end and the full autoglucosylated enzyme continued catalytically active for transglucosylation of the alternative substrate dodecyl-maltose. Even the autoglucosylation cessation once glycogenin acquired a mature maltosaccharide moiety, proteoglycogen and glycogenin species ranging rM 47-200 kDa, derived from proteoglycogen, showed to be autoglucosylable. The results describe for the first time the ability of polysaccharide-bound glycogenin for intramolecular autoglucosylation, providing evidence for cessation of the glucose polymerization initiated into the tyrosine residue, by inaccessibility of the acquired maltosaccharide moiety to further autoglucosylation.     

Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle

Purified preparations of glycogen synthase are a complex of two proteins, the catalytic subunit of glycogen synthase and glycogenin, present in a 1:1 molar ratio [J. Pitcher, C. Smythe, D. G. Campbell & P. Cohen (1987) Eur. J. Biochem. 169, 497-502]. This complex has now been found to contain a further glucosyltransferase activity that catalyses the transfer of glucose residues from UDP-Glc to glucosylated-glycogenin. The glucosyltransferase, which is of critical importance in forming the primer required for de novo glycogen biosynthesis, is distinct from glycogen synthase in several ways. It has an absolute requirement for divalent cations, a 1000-fold lower Km for UDP-Glc and its activity is unaffected by incubation with UDP-pyridoxal or exposure to 2 M LiBr, which inactivate glycogen synthase by 95% and 100%, respectively. The priming glucosyltransferase and glycogen synthase activities coelute on Superose 6, and the rate of glycosylation of glycogenin is independent of enzyme concentration, suggesting that the reaction is catalysed intramolecularly by a subunit of the glycogen synthase complex. This component has been identified as glycogenin, following dissociation of the subunits in 2 M LiBr and their separation on Superose 12. The glycosylation of isolated glycogenin reaches a plateau when five additional glucose residues have been added to the protein, and digestion with alpha-amylase indicates that all the glycogenin molecules contain at least one glucosyl residue prior to autoglucosylation. The priming glucosyltransferase activity of glycogenin is unaffected by either glucose 6-phosphate or by phosphorylation of the catalytic subunit of glycogen synthase. The mechanism of primer formation is discussed in the light of the finding that glycogenin is an enzyme that catalyses its own autoglucosylation.     

Structural and functional studies on rabbit liver glycogenin

Glycogenin, the protein primer required for the biogenesis of muscle glycogen, has been isolated from rabbit liver glycogen. The protein comprised 0.0025% of liver glycogen by mass, 200-fold lower than the glycogenin content of muscle glycogen. Structural analyses, including determination of the amino acid sequence surrounding the glucosylated-tyrosine residue, showed identity with muscle glycogenin. Catalytically active liver glycogenin was partially purified and, like the skeletal muscle protein, catalysed an intramolecular, Mn2+- and UDP-Glc-dependent autoglucosylation reaction, forming a primer on which glycogen synthase could act. The results demonstrate that hepatic and muscle glycogenins are almost certainly identical proteins and that liver and skeletal muscle share a common mechanism for the biogenesis of glycogen molecules. The results also indicate that there is about one glycogenin molecule/liver glycogen alpha particle.     

C-chain-bound glycogenin is released from proteoglycogen by isoamylase and is able to autoglucosylate

Proteoglycogen glycogenin is linked to the glucose residue of the C-chain reducing end of glycogen. We describe for the first time the release by isoamylase and isolation of C-chain-bound glycogenin (C-glycogenin) from proteoglycogen. The treatment of proteoglycogen with alpha-amylase releases monoglucosylated and diglucosylated glycogenin (a-glycogenin) which is able to autoglucosylate. It had been described that isoamylase splits the glucose-glycogenin linkage of fully autoglucosylated glycogenin previously digested with trypsin, releasing the maltosaccharide moiety. It was also described that carbohydrate-free apo-glycogenin shows higher mobility in SDS-PAGE and twice the autoglucosylation capacity of partly glucosylated glycogenin. On the contrary, we found that the C-glycogenin released from proteoglycogen by isoamylolysis shows lower mobility in SDS-PAGE and about half the autoglucosylation acceptor capacity of the partly glucosylated a-glycogenin. This behavior is consistent with the release of maltosaccharide-bound glycogenin instead of apo-glycogenin. No label was split from auto-[14C]glucosylated C-glycogenin or fully auto-[14C]glucosylated a-glycogenin subjected to isoamylolysis without previous trypsinolysis, thus proving no hydrolysis of the maltosaccharide-tyrosine linkage. The ability of C-glycogenin for autoglucosylation would indicate that the size of the C-chain is lower than the average length of the other glycogen chains.     

Isolation and structural analysis of a peptide containing the novel tyrosyl-glucose linkage in glycogenin.

The glucosylation site on glycogenin, the protein primer required for de novo glycogen synthesis, has been identified. The glucose is attached at position C1 in a glycosidic linkage with a unique tyrosine, and the sequence surrounding this residue was found to be: His-Leu-Pro-Phe-Ile-Tyr-Asn-Leu-Ser-Ser-Ile-Ser-Ile-Tyr(Glc)-Ser-Tyr-Leu -Pro- Ala-Phe-Lys. The same tyrosine residue is glycosylated whether glycogenin is isolated as a complex with the catalytic subunit of glycogen synthase, or covalently attached to glycogen. The possibility that insulin and growth factors may enhance glycogen synthesis via stimulation of the priming reaction is discussed.     

Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin

Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.     

Direct detection of glycogenin reaction products during glycogen initiation

Glycogenin initiates glycogen synthesis in an autocatalytic reaction in which individual glucose residues are covalently linked to Tyrosine 194 in order to form a short priming chain of glucose residues that is a substrate for glycogen synthase which, combined with the branching enzyme, catalyzes the bulk synthesis of glycogen. We sought to develop a new enzymatic assay to better characterize both the chemical and enzymatic characteristics of this unusual reaction. By directly detecting the reaction products using electrospray mass spectrometry this procedure permits both the visualization of the intact individual reaction species produced as a function of time and quantitation of the levels of each of species. The quantitation of the reaction agrees well with previous measurements of both catalytic rate and the change in rate as a function of average glucosylation. The results from this assay provide new insight into the mechanism by which glycogenin catalyzes the initiation reaction.     

Structural and biochemical insight into glycogenin inactivation by the glycogenosis-causing T82M mutation

The X-ray structure of rabbit glycogenin containing the T82M (T83M according to previous authors amino acid numbering) mutation causing glycogenosis showed the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP-glucose hydrolyzing activities were abolished even though affinity and interactions with UDP-glucose and positioning of Tyr194 acceptor were conserved. Substitution of Thr82 for serine but not for valine restored the maximum extent of autoglucosylation as well as transglucosylation and UDP-glucose hydrolysis rate. Results provided evidence sustaining the essential role of the lost single hydrogen bond for UDP-glucose activation leading to glycogenin-bound glycogen primer synthesis.     

Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction

Glycogenin is a dimeric self-glucosylating protein involved in the initiation phase of glycogen biosynthesis. As an enzyme, glycogenin has the unusual property of transferring glucose residues from UDP-glucose to itself, forming an alpha-1,4-glycan of around 10 residues attached to Tyr194. Whether this self-glucosylation reaction is inter- or intramolecular has been debated. We used site-directed mutagenesis of recombinant rabbit muscle glycogenin-1 to address this question. Mutation of highly conserved Lys85 to Gln generated a glycogenin mutant (K85Q) that had only 1-2% of the self-glucosylating activity of wild-type enzyme. Consistent with previous work, mutation of Tyr194 to Phe in a GST-fusion protein yielded a mutant, Y194F, that was catalytically active but incapable of self-glucosylation. The Y194F mutant was able to glucosylate the K85Q mutant. However, there was an initial lag in the self-glucosylation reaction that was abolished by preincubation of the two mutant proteins. The interaction between glycogenin subunits was relatively weak, with a dissociation constant inferred from kinetic experiments of around 2 microM. We propose a model for the glucosylation of K85Q by Y194F in which mixing of the proteins is followed by rate-limiting formation of a species containing both subunit types. The results provide the most direct evidence to date that the self-glucosylation of glycogenin involves an inter-subunit reaction.     

A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis

In this paper we elucidate part of the mechanism of the early stages of the biosynthesis of glycogen. This macromolecule is constructed by covalent apposition of glucose units to a protein, glycogenin, which remains covalently attached to the mature glycogen molecule. We have now isolated, in a 3500-fold purification, a protein from rabbit muscle that has the same Mr as glycogenin, is immunologically similar, and proves to be a self-glucosylating protein (SGP). When incubated with UDP-[14C]glucose, an average of one molecular proportion of glucose is incorporated into the protein, which we conclude is the same as glycogenin isolated from native glycogen. The native SGP appears to exist as a high-molecular-weight species that contains many identical subunits. Because the glucose that is self-incorporated can be released almost completely from the acceptor by glycogenolytic enzymes, the indication is that it was added to a preformed chain or chains of 1,4-linked alpha-glucose residues. This implies that SGP already carries an existing maltosaccharide chain or chains to which the glucose is added, rather than glucose being added directly to protein. The putative role of SGP in glycogen synthesis is confirmed by the fact that glucosylated SGP acts as a primer for glycogen synthase and branching enzyme to form high-molecular-weight material. SGP itself is completely free from glycogen synthase. The quantity of SGP in muscle is calculated to be about one-half the amount of glycogenin bound in glycogen.     

Conformational analysis of intermediates involved in the in vitro folding pathways of recombinant human macrophage colony stimulating factor beta by sulfhydryl group trapping and hydrogen/deuterium pulsed labeling

In vitro oxidative folding of reduced recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) involves two major events: disulfide isomerization in the monomeric intermediates and disulfide-mediated dimerization. Kinetic analysis of rhm-CSFbeta folding indicated that monomer isomerization is slower than dimerization and is, in fact, the rate-determining step. A time-dependent determination of the number of free cysteines remaining was made after refolding commence. The folding intermediates revealed that rhm-CSFbeta folds systematically, forming disulfide bonds via multiple pathways. Mass spectrometric evidence indicates that native as well as non-native intrasubunit disulfide bonds form in monomeric intermediates. Initial dimerization is assumed to involve formation of disulfide bonds, Cys 157/159-Cys' 157/159. Among six intrasubunit disulfide bonds, Cys 48-Cys 139 and Cys' 48-Cys' 139 are assumed to be the last to form, while Cys 31-Cys' 31 is the last intersubunit disulfide bond that forms. Conformational properties of the folding intermediates were probed by H/D exchange pulsed labeling, which showed the coexistence of noncompact dimeric and monomeric species at early stages of folding. As renaturation progresses, the noncompact dimer undergoes significant structural rearrangement, forming a native-like dimer while the monomer maintains a noncompact conformation.     

GNN is a self-glucosylating protein involved in the initiation step of glycogen biosynthesis in Neurospora crassa

The initiation of glycogen synthesis requires the protein glycogenin, which incorporates glucose residues through a self-glucosylation reaction, and then acts as substrate for chain elongation by glycogen synthase and branching enzyme. Numerous sequences of glycogenin-like proteins are available in the databases but the enzymes from mammalian skeletal muscle and from Saccharomyces cerevisiae are the best characterized. We report the isolation of a cDNA from the fungus Neurospora crassa, which encodes a protein, GNN, which has properties characteristic of glycogenin. The protein is one of the largest glycogenins but shares several conserved domains common to other family members. Recombinant GNN produced in Escherichia coli was able to incorporate glucose in a self-glucosylation reaction, to trans-glucosylate exogenous substrates, and to act as substrate for chain elongation by glycogen synthase. Recombinant protein was sensitive to C-terminal proteolysis, leading to stable species of around 31kDa, which maintained all functional properties. The role of GNN as an initiator of glycogen metabolism was confirmed by its ability to complement the glycogen deficiency of a S. cerevisiae strain (glg1 glg2) lacking glycogenin and unable to accumulate glycogen. Disruption of the gnn gene of N. crassa by repeat induced point mutation (RIP) resulted in a strain that was unable to synthesize glycogen, even though the glycogen synthase activity was unchanged. Northern blot analysis showed that the gnn gene was induced during vegetative growth and was repressed upon carbon starvation.     

The discovery of glycogenin and the priming mechanism for glycogen biogenesis

The biogenesis of glycogen in skeletal muscle requires a priming mechanism that has recently been elucidated. The first step is catalysed by a protein tyrosine glucosyltransferase and involves the formation of a novel glycosidic linkage, namely the covalent attachment of glucose to a single tyrosine residue (Tyr194) on a priming protein, termed glycogenin. The next stage is the extension of the glucan chain from Tyr194 and involves the sequential addition of up to seven further glucosyl residues. This reaction is brought about autocatalytically by glycogenin itself, which is a Mn2+/Mg(2+)-dependent UDP-Glc-requiring glucosyltransferase. The glucan primer is elongated by glycogen synthase, but only when glycogenin and glycogen synthase are complexed together. Glycogen synthase dissociates from glycogenin during the synthesis of a glycogen molecule, enabling glycogen molecules to reach their maximum theoretical size. Each mature glycogen beta particle in muscle contains one molecule of glycogenin attached covalently, and an average one glycogen synthase catalytic subunit bound non-covalently. As evidence accumulates that a priming protein may be a fundamental property of polysaccharide synthesis in general, the molecular details of mammalian glycogen biogenesis may serve as a useful model for other systems.     

Proglycogen: a low-molecular-weight form of muscle glycogen.

We recently reported that muscle contains a trichloroacetic acid-precipitable component having Mr approx. 400 kDa that can be glucosylated by an endogenous enzyme acting on UDPglucose. This component contains within itself the autocatalytic, self-glucosylating protein glycogenin, the primer for glycogen synthesis. We now report that this substance, to which we give the name proglycogen, is a glycogen-like molecule constituting about 15% of total glycogen. It acts as a very efficient acceptor of glucose residues added from UDPglucose. Further, that the endogenous enzyme that adds the glucose to proglycogen is not the autocatalytic protein but a glycogen synthase-like enzyme. Proglycogen may be an intermediate in the synthesis and degradation of macromolecular glycogen and may exist and be metabolized as a separate entity. Consideration should now be given to the revival of the concept that tissue contains two forms of glycogen. One is proglycogen. The other is the 'classical', macromolecular glycogen. Additionally, proglycogen and glycogen may be glucosylated by different forms of synthase.     

Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration

The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by alpha-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences (P < 0.05) between HL and LL for total glycogen (438.0 +/- 69.5 vs. 305.7 +/- 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 +/- 38.1 vs. 227.3 +/- 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity (r = 0.84, P < 0.001) was observed. Similar positive correlations (P < 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal muscle.     

Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.     

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 °C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant k _dcalculated for the monomer at 85 °C was 0.029 min^-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 °C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 °C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.     

Dodecyl-{beta}-D-maltoside as a substrate for glucosyl and xylosyl transfer by glycogenin

Glycogenin is the core protein of glycogen proteoglycan andis, at the same time, a self-glucosylating enzyme which catalysesearly glucosyl transfer steps in the biosynthesis of glycogen.In the course of this process, glycogenin is glucosylated progressivelyuntil an oligosaccharide containing 8–11 glucose residueshas been formed. Although glycogenin, under physiological conditions,is both enzyme and acceptor in the glucosyl transfer reactions,it is also capable of utilizing p-nitrophenyl-linked malto-oligosaccharidesas exogenous acceptors. In view of the potential usefulnessof exogenous acceptors in the study of the mechanism of theglycogenin reaction, we have expanded the search for such compoundsand report here that several alkyl glucosides and alkyl maltosidesmay serve as acceptors in glucosyl transfer by beef kidney glycogenin.Dodecyl-ß-D-maltoside (K_m {small tilde}100 µM)was the most effective acceptor among the compounds tested andyielded 30 times as much product as p-nitrophenyl-     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.