Gisèle Préaux

THE QUATERNARY STRUCTURE OF SEPIA-OFFICINALIS HEMOCYANIN

Abstract The haemocyanin (Hc) of Sepia officinalis is constituted of ten identical subunits ( M r about 390 000), which consist of eight functional (dioxygen-binding) units, designated by the letters a–h . A model is proposed for the quaternary structure of this HC, based on immuno-electron microscopy of whole molecules and on morphological studies of compact 1/5 molecules (consisting of two associated subunits). The immuno-electron microscopy was performed with IgG fractions containing antibodies specific for fragments abc, gh and de , respectively. Based on their general appearance, the immunocomplexes were divided into twelve types, whose occurernce was determined for the three IgG fractions. For each type of complex the location of the attachment sites of the IgGs was estimated in the cylindrical Hc molecule subdivided for this purpose into three regions (an upper, middle and lower third). The results indicate that functional units b to g form the wall of the cylindrical Hc molecule, while the two outer functional units of a and h are folded inside, thus forming a collar at the top and bottom of the cylinder. An experiment with a mixture of IgG fractions anti- abc and anti- gh showed that the ten subunits ( a–h ) lie parallel to each other, giving rise to an asymmetric molecule with, however, a symmetric appearance in the electron microscope.

Influence of the protein conformation on the interaction between α-lactalbumin and dimyristoylphosphatidylcholine vesicles

alpha-Lactalbumin is a globular protein containing helical regions with highly amphiphathic character. In this work, the interaction between bovine alpha-lactalbumin and sonicated dimyristoylphosphatidylcholine vesicles has been compared in different circumstances which influence the protein conformation i.e., pH, ionic strength, decalcification, guanidine hydrochloride denaturation. Above the isoelectric point the interaction is mainly electrostatic; improved electrostatic interaction results in better contact with the apolar lipid phase. Below the isoelectric point, hydrophobic forces dominate the interaction and the vesicles are solubilized. The mode of interaction is not determined to a great extent by the demetallization of the protein. However, by a more explicit unfolding of the globular structure with guanidine hydrochloride, micellar complexes can be formed with the lipid, even at neutral pH. From this study it is obvious that the presence or capability for formation of helices with high amphipathic character is not a sufficient condition for lipid solubilization by a globular protein. Also, the capability of a globular protein to unfold its tertiary structure seems to be a prerequisite for its capability to lipid solubilization.

Relative molecular mass of the polypeptide chain of βc-haemocyanin of Helix pomatia and carbohydrate composition of the functional units

1. 1. Sedimentation equilibrium at pH 9.25 of the subunit (twentieth molecules) of βc-haemocyanin of Helix pomatia at protein concentrations below 0.6 mg/ml yielded a Mw value of ⋍4.5 × 105, in good agreement with the Mr value (9 × 106) of the whole molecule. 2. 2. The sum of the Mr values of the tryptic fragments from sedimentation equilibrium and of the eight functional units from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), amounted to 4.6 × 105. 3. 3. Carbohydrate is unevenly distributed over the functional units: g >b >e >a >h >d >f >c. On subtracting its weight contribution (8.25%) a Mr of ⋍4.2 × 105 was obtained for the polypeptide part of the subunit. 4. 4. The βc-haemocyanin subunit and some of its proteolytic fragments are proposed as markers for SDS-PAGE in the high-Mr range.

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 μg per style, which was 40–60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S1-, S2- and S3-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.

Presence of Only Seven Functional Units in the Polypeptide Chain of the Haemocyanin of the Cephalopod Octopus Vulgaris

The subunits of the haemocyanins (Hc’s) of gastropods (e.g. Helix pomatia, M r ≃ 450 000) and of decapodan cephalopods (e.g. Sepia officinalis, M r ≃ 390 000) are constituted of eight functional units (each containing a dioxygen-binding copper pair) with average M r of respectively ≃ 55 000 and ≃ 50 000 (1). In order to check if the Hc subunits of octopodan cephalopods are built in the same way, the subunits of Octopus vulgaris Hc were submitted to limited proteolysis.

Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment.

A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.

Gel chromatographic separation of the haemocyanins of Helix pomatia. Further electrophoretic and immunological characterization of the components

Abstract 1. 1. Chromatography on agarose gels at pH 5.5 in the presence of 1 M NaCI allowed to separate the total β-haemocyanin(β-Hc) of Helix pomatia from the α-haemocyanin (α-Hc). 2. 2. When crystalline β-Hc (β c -Hc) was first insolubilized by dialysis at pH 5.3 at low ionic strength the chromatographic separation yielded the soluble β-haemocyanin fraction (β 3 -Hc) and α-Hc. 3. 3. The amount of β c -Hc varied from one third to one half of β-Hc with the lowest value for non-hibernating snails. 4. 4. β 2 -Hc, in contrast to β c -Hc, did not differ from α-Hc in its electrophoretic and immunological properties.

Structural Investigations on β-Haemocyanin of Helix pomatia by Limited Proteolysis

The haemocyanin of the Roman snail (Helix pomatia) consists of an α- (75%) and a β-component (25%), both having a molecular weight of 9 × 106 (Wood et al., 1971) and a copper content of the order of 0.25% (2 Cu/ 50,000 on the average). The α-component dissociates into halves in the presence of 1 M NaCl or KC1 (Heirwegh et al., 1961) or in slightly alkaline medium, pH 7.0–7.6 (Witters and Lontie, 1968), while the β- component does not. At higher pH values both haemocyanins successively dissociate into tenths and twentieths (Witters and Lontie, 1968; Siezen and van Driel, 1974). As the twentieths do not dissociate further under mild conditions, they most probably correspond to the polypeptide chains of the haemocyanin molecule.

Involvement of glycans in the immunological cross-reaction between α-macroglobulin and hemocyanin of the gastropod Helix pomatia

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between alpha-macroglobulin (alphaM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: alphaM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: alpha(D)-HpH, alpha(N)-HpH and beta-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With beta-HpH, which is richer in carbohydrates than alpha(D)-HpH and alpha(N)-HpH, mainly due to a higher 3-O-methyl-d-galactose content, the cross-reaction with HpalphaM was highest. (ii) From the 8 functional units, designated a-h, isolated from beta-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against HpalphaM, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both beta-HpH and HpalphaM are further evidence that glycans are involved in the immunological relationship between HpH and HpalphaM. Carbohydrate analyses indicated that the glycan structures present on HpalphaM are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3-O-methyl-d-galactose seem to be responsible for the cross-reactivity since the alpha-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.

Identification, separation and characterization of the haemocyanin components of Helix aspersa

Abstract 1. 1. The haemocyanin (Hc) of Helix aspersa , like that of Helix pomatia , consists of β-Hc, α D -Hc and α N -Hc. These components were separated by chromatography and isoelectric fractination. 2. 2. The β-Hc content of nonhibernating snails from Belgium was 18–19% against 10% for those from Tunisia with a concomitant increase of α D -Hc. 3. 3. The pI of the α-Hcs amounted to 4.6 against 5.3 for H. pomatia , that of β-Hc to 5.2 against 5.4. 4. 4. Dialysis at pH 5.2 and low I yielded mixed crystals (60% β-Hc, 40% α D -Hc). 5. 5. The alkaline dissociation of the Hc components was similar to those of H. pomatia but with a stronger stabilization of the half molecules of α D -Hc.