Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the α- and β-subunits of (Na⁺ + K⁺)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[¹²⁵I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na⁺ + K⁺)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na⁺ + K⁺)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of ¹²⁵I-labeling within the α-subunit. The preservation of (Na⁺ + K⁺)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E₁- and E₂-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Tryptic digest of the α subunit of lamb kidney (Na+ + K+)-ATPase

The M_r ≈ 100 000 α subunit was prepared from highly purified lamb kidney (Na⁺+ K⁺)-ATPase. Its N-terminal sequence is Gly-Arg-Asx-Lys-Tyr-Glu. The α subunit was S-carboxymethylated, succinylated, and cleaved at its 40 arginine residues with trypsin. Four major, well-differentiated peptide fractions (A to D) were obtained by chromatography of the digest on a Sephadex G-50 column. Fraction A eluted at the void volume of the column and contained aggregated, very hydrophobic peptides, possibly from regions of α that are buried within the membrane lipid bilayer in the native enzyme. Fractions B to D, which together accounted for about 75% of the total protein, contained water-soluble peptides. To test the feasibility of using antibodies to identify and purify specific peptides of α subunit, studies were carried out using antibodies to native (Na⁺+ K⁺)-ATPase. Carboxymethylation and succinylation did not significantly decrease total antibody binding to α subunit, although the affinity of the anti-(Na⁺ + K⁺)-ATPase antibodies for α subunit was reduced by about 50%. The tryptic peptides of a subunit also retain significant immunochemical reactivity. Fractions A, B and C (but not D) of the digest all bind antibodies. To characterize further the tryptic digest, 16 peptides from fraction D were isolated and sequence studies on these were carried out.     

Characterization of (Na+ + K+)-ATPase liposomes: II. Effect of α-subunit digestion on intramembrane particle formation and Na+,K+-transport

The effect of the protein structure of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ on its incorporation into liposome membranes was investigated as follows: the catalytic α-subunit of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ decrease progessively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight α-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with intact protein structure and digested, non functional enzyme consisting of fragments of the α-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

Allosteric property of the (Na⁺+K⁺)-ATPase β₁ subunit

(Na(+)+K(+))-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na(+) and K(+) ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β(1) subunit can generate a significant impact on the catalytic function of NKA. By using a β(1) subunit-specific monoclonal antibody JY2948, we found that the JY2948-β(1) subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na(+) and K(+) ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β(1) subunit and uncovers the latent allosteric property of the β(1) subunit, which remotely controls the NKA catalytic function.     

Expression of Na+/K+-ATPase α-subunit mRNA during embryonic development of the crayfish Astacus leptodactylus

Astacus leptodactylus is a decapod crustacean fully adapted to freshwater where it spends its entire life cycle after hatching under huge osmoconcentration differences between the hemolymph and surrounding freshwater. We investigated the expression of mRNA encoding one ion transport-related protein, Na⁺/K⁺-ATPase α-subunit, and one putative housekeeping gene, β-actin, during crayfish ontogenesis using quantitative real-time PCR. A 216-amino acid part of the open reading frame region of the cDNA coding for the Na⁺/K⁺-ATPase α-subunit was sequenced from total embryo, juvenile and adult gill tissues. The predicted amino acid sequence showed a high percentage similarity to those of other invertebrates (up to 95%) and vertebrates (up to 69%). β-actin expression exhibited modest changes through embryonic development and early post-embryonic stage. The Na⁺/K⁺-ATPase α-subunit gene was expressed in all studied stages from metanauplius to juvenile. Two peaks of expression were observed: one in young embryos at 25% of embryonic development (EI = 100 μm), and one in embryos just before hatching (at EI = 420 μm), continuing in the freshly hatched juveniles. The Na⁺/K⁺-ATPase expression profile during embryonic development is time-correlated with the occurrence of other features, including ontogenesis of excretory antennal glands and differentiation of gill ionocytes linked to hyperosmoregulation processes and therefore involved in freshwater adaptation.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Allosteric property of the (Na+K)-ATPase β1 subunit

(Na⁺+K⁺)-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na⁺ and K⁺ ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β₁ subunit can generate a significant impact on the catalytic function of NKA. By using a β₁ subunit-specific monoclonal antibody JY2948, we found that the JY2948–β₁ subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na⁺ and K⁺ ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β₁ subunit and uncovers the latent allosteric property of the β₁ subunit, which remotely controls the NKA catalytic function.     

Identification of an intra-molecular disulfide bond in the sodium channel β1-subunit

The sodium channel β1 subunit is non-covalently associated with the pore-forming α-subunits, and has been proposed to act as a modulator of channel activity, regulator of channel cell surface expression and cell adhesion molecule. Its importance is evident since mutations of the β1 subunit cause neurologic and cardiovascular disorders. The first described β1 subunit mutation is the C121W, that is related to generalized epilepsy with febrile seizures plus (GEFS+), a childhood genetic epilepsy syndrome. This mutation changed a conserved cysteine residue in position 121 into a tryptophan, putatively disrupting a disulfide bridge that should normally maintain the β1 extracellular immunoglobulin-like fold. Using the 2-D-diagonal-SDS-PAGE technique, we demonstrated the existence of this putative disulfide bridge in the Ig-like extracellular domain of the β1 subunit and its disruption in the epileptogenic C121W mutant.     

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β-subunit by disruption of disulfide bonds

The human chorionic gonadotropin β-subunit (hCGβ) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGβ produced in Chinese hamster ovary (CHO) cells: wild-type hCGβ (βWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of βWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled βWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGβ molecule. Abbreviations: hCGβ, human chorionic gonadotropin β-subunit; βWT, wild type hCGβ; CHO, Chinese hamster ovary; Endo-H, endoglycosidase H; Endo-F, endoglycosidase F This revised version was published online in November 2006 with corrections to the Cover Date.     

Erythrocyte Membrane Digoxin-Sensitive (Na+–K+)-ATPase of Non-Insulin Dependent Diabetic Humans

Erythrocyte plasma membranes of non-insulin dependent diabetic humans (NIDDM) and healthy humans were prepared by hypotonic lysis. The specific activity of (Na⁺–K⁺)-ATPase of NIDDM membranes, both in the absence and presence of digoxin were lower than the specific activity of normal enzymes (83.6 percent and 74.0 percent of the normal enzyme respectively). Addition of digoxin decreased the activity of this enzyme (38.0 percent in NIDDM and 30.0 percent in normal enzyme).Although the affinity of the pump for ATP was similar in both membranes of NIDDM and normal humans (K_m for ATP=19.9±0.24M ATP and 20.0±0.21 M ATP respectively), the V_max of NIDDM membranes was more than 20 percent lower than that of the normal enzyme. The specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺–Mg²⁺)-ATPase) of NIDDM membrane was lower than 80 percent of the specific activity of the normal enzymes. While the affinity of the pump for ATP was lower in the membranes of NIDDM (K_m for ATP=50.0±4.3 M ATP) in comparison to normal membranes (K_m for ATP=63.1±38M ATP), the V_max of NIDDM membranes was similar to the normal enzyme. Altogether, these findings suggest that both the (Na⁺–K⁺)-ATPase and Ca²⁺-pumping ATPase of NIDDM membranes are less functional than the enzymes in normal erythrocytes.     

The expression of Na+/K+-ATPase β-subunit cRNA injected into Xenopus oocytes is affected by coinjection with α-subunit cRNA

The cRNA for Torpedo californica Na⁺/K⁺-ATPase -subunit (cRNA) was injected into Xenopus oocytes alone or with the cRNA for the Na⁺/K⁺-ATPase -subunit (cRNA). When cRNA was injected alone, the amount of the -subunit that accumulated in oocytes increased with increasing amounts of injected cRNA. When cRNA and cRNA were injected simultaneously, less -subunit accumulated than when cRNA was injected alone, whereas the Na⁺/K⁺-ATPase activity increased markedly. The decrease in the accumulation of the -subunit was dose-dependent upon the cRNA. The mutant -subunit unable to assemble with the -subunit accumulated in oocytes independently of cRNA, suggesting that post-translational control mechanisms may serve to reduce the accumulation of the -subunit.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 05259226, No. 06454149).     

Characteristics of antibody inhibition of rat kidney (Na+−K+)-ATPase

Summary Antibodies which were raised against highly purified membrane-bound (Na⁺–K⁺)-ATPase from the outer medulla of rat kidneys inhibit the (Na⁺–K⁺)-ATPase activity up to 95%. The antibody inhibition is reversible. The time course of enzyme inhibition and reactivation is biphasic in semilogarithmic plots.In the purified membrane-bound (Na⁺–K⁺)-ATPase negative cooperativity was observed (a) for the ATP dependence of the (Na⁺–K⁺)-ATPase activity (n=0.86), (b) for the ATP binding to the enzyme (n=0.58), and (c) for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity (n=0.77). By measuring the Na⁺ dependence of the (Na⁺–K⁺-ATPase reaction, a positive homotropic cooperativity (n=1.67) was found.As reactivation of the antibody-inhibited enzyme proceeds very slowly (t _0.5=5.2hr), it was possible to measure characteristics of the antibody-(Na⁺–K⁺)-ATPase complex: The antibodies exerted similar effects on the ATP dependence of the (Na⁺–K⁺)-ATPase reaction and on the ATP binding of the enzyme.V _max of the (Na⁺–K⁺)-ATPase reaction and the number of ATP binding sites were reduced whileK _{0.5 ATP} for the (Na⁺–K⁺)-ATPase activity and for the ATP binding were increased by the antibodies. The Hill coefficients for the ATP binding and for the ATP dependence of the enzyme activity were not significantly altered by the antibodies. The antibodies increased theK _0.5 value for the Na⁺ stimulation of the (Na⁺–K⁺)-ATPase activity, but they did not alter the homotropic interactions between the Na⁺-binding sites. The negative cooperativity which was observed for the ouabain inhibition of the (Na⁺–K⁺)-ATPase activity was abolished by the antibodies.The data are tentatively explained by the following model: The antibodies bind to the (Na⁺–K⁺)-ATPase from the inner membrane side, reduce the ATP binding symmetrically at the ATP binding sites and reduce thereby also the (Na⁺–K⁺)-ATPase activity of the enzyme. The antibodies may inhibit the ATP binding by a direct interaction or by means of a conformational change at the ATP binding sites. This may possibly also lead to the alteration of the Na⁺ dependence of the (Na⁺–K⁺)-ATPase activity and to the observed alteration of the dose response to the ouabain inhibition.     

Serine496 of β2 subunit of L-type Ca2+ channel participates in molecular crosstalk between activation of (Na++K+)-ATPase and the channel

Activation of (Na++K+)-ATPase (NKA) regulates cardiac L-type Ca2+ channel (LTCC) function through molecular crosstalk. The mechanism underlying NKA-LTCC crosstalk remains poorly understood. We have previously shown that activation of NKA leads to phosphorylation of LTCC α1 Ser1928. Here we investigated whether LTCC β2 subunit is modulated by NKA activation and found that LTCC β2 Ser496 is phosphorylated in response to activation of NKA. Src inhibitor PP1 and Erk1/2 inhibitor PD98059 abolish LTCC β2 Ser496 phosphorylation, suggesting that NKA-mediated β2 Ser496 phosphorylation is dependent of Src/Erk1/2 signaling pathway. Protein kinase G (PKG) inhibitor KT5823 failed to inhibit the phosphorylation of β2 Ser496, indicating that the NKA-LTCC crosstalk is independent of PKG activity. The results of nifedipine sensitive 45Ca influx experiments suggest that phosphorylation of β2 Ser496 may play a key down-regulation role in attenuating the accelerated activity of α1 subunit of the channel. Ouabain does not cause a phosphorylation on β2 Ser496, indicating a fundamental difference between activation and inhibition of NKA-mediated biological processes. This study provides the first evidence to demonstrate that LTCC β2 subunit is coupled with the movement of signals in the mechanism of activation of NKA-mediated crosstalk with LTCC.     

Stimulation of (Na+K+)-ATPase of rat liver plasma membrane by amino acids

The effect of twelve l-amino acids on the activity of liver plasma membrane (Na⁺K⁺)-ATPase has been tested. Histidine and arginine significantly enhanced the activity. The activtion by histidine showed saturation kinetics with an apparent K_a of about 8 mM, and was evident over a wide range of Na⁺ concentrations. The same amino acid did not significantly affect the Mg²⁺-dependent ATPase activity.     

The sided action of Na+ and of K+ on reconstituted shark (Na+ + K+)-ATPase engaged in Na+−Na+ exchange accompanied by ATP hydrolysis. I. The ATP activation curve

The ATP hydrolysis dependent Na⁺-Na⁺ exchange of reconstituted shark (Na⁺ + K⁺)-ATPase is electrogenic with a transport stoichiometry as for the Na⁺-K⁺ exchange, suggesting that translocation of extracellular Na⁺ is taking place via the same route as extracellular K⁺. The preparation thus offers an opportunity to compare the sided action of Na⁺ and of K⁺ on the affinity for ATP in a reaction in which the intermediary steps in the overall reaction seems to be the same without and with K⁺. With Na⁺ but no K⁺ on the two sides of the enzyme, the ATP-activation curve is hyperbolic and the affinity for ATP is high. Extracellular K⁺ in concentrations of 50 μM (the lowest tested) and up gives biphasic ATP activation curves, with both a high- and a low-affinity component for ATP. Cytoplasmic K⁺ also gives biphasic ATP-activation curves, however, only when the K⁺ concentration is 50 mM or higher (Na⁺ + K⁺ = 130 mM). The different ATP-activation curves are explained from the Albers-Post scheme, in which there is an ATP-dependent and an ATP-independent deocclusion of E₂(Na₂⁺) and E₂(K₂⁺), respectively, and in which the dephosphorylation of E₂-P is rate limiting in the presence of Na⁺ (but no K⁺) extracellular, whereas in the presence of extracellular K⁺ it is the deocclusion of E₂(K₂⁺) which is rate limiting.     

Na+-transport modulation induces isoform-specific expression of Na+,K+-ATPase α-subunit isoforms in C2C12 skeletal muscle cell

Changes in demands for Na⁺ transport alter expression of the Na⁺,K⁺-ATPase subunit isoforms. In skeletal muscle, the effects of these changes on expression the 2 isoform, the major isoform expressed in differentiated muscle cell, is not known. Therefore, this study examines regulation of the -subunit isoforms by Na⁺ in the C₂C₁₂ skeletal muscle cell that expresses the 1 and 2 isoforms. Western blot analysis showed that in differentiating C₂C₁₂ muscle cell, but not in undifferentiated myoblast, veratridine, a Na⁺ channel activator, greatly increased expression of the 2 isoform; expression of 1 was unaltered. Because the level of -actinin was unaltered, the data suggest that veratridine treatment did not significantly alter the progression of cell differentiation. Furthermore, a reduction in Na⁺ transport by tetrodotoxin again failed to alter expression of a1. Thus, in C₂C₁₂ skeletal muscle cell, changes in Na⁺ transport alters expression of the 2, but not the 1 isoform. These results differ from those observed previously in muscle cells that express only the 1 isoform. Because mammalian skeletal muscle expresses both the 1- and 2-subunit isoforms, the differential regulation that was observed may be physiologically relevant in these muscle cells in vivo.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.