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.

Primary structure of 21 novel monoantennary and diantennary N-linked carbohydrate chains from alphaD-hemocyanin of Helix pomatia.

The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein alphaD-hemocyanin (alphaD-Hc) of Helix pomatia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an alphaD-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH2, was characterized using 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of alphaD-Hc were released with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH2/Lichrosphere-NH2, and/or Lichrosphere-C18. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 1H-NMR spectroscopy. In total, four novel types of antenna were identified, namely, [structures: see text] which are all attached to O-2 of alphaMan residues of the trimannosyl-N,N'-diacetylchitobiose core element, which is generally beta-1,2-xylosylated and alpha-1,6-fucosylated, Man(alpha1-6)[Man(alpha1-3)][+/-Xyl(beta1-2)]Man(beta1-4)GlcNAc(beta1-4) [+/-Fuc(alpha1-6)]GlcNAc.

Evidence for a cysteine-histidine thioether bridge in functional units of molluscan haemocyanins and location of the disulfide bridges in functional units d and g of the betaC-haemocyanin of Helix pomatia.

In functional units d and g from the betaC-haemocyanin of the gastropod Helix pomatia, aminoacid analysis in the presence of dimethyl sulfoxide showed the occurrence of seven cysteine residues. Titration with 5,5'-dithiobis(2-nitrobenzoic acid), however, did not reveal any free thiol group. Pepsinolysis at pH 2.0 followed by amino acid analysis and partial sequencing of the cysteine-containing peptides showed that six cysteine residues are involved in the formation of three disulfide bridges at corresponding positions. The results indicated that the remaining cysteine residue is linked by a thioether bridge to a histidine residue located two positions further in the sequence (H. pomatia d Cys60 His62; H. pomatia g Cys66 His68). This residue corresponds to one of the three histidine residues considered to be involved in the coordination of the copper A atom of the dinuclear copper group of the functional units. The presence of the thioether bond was further evidenced by an absorption band at 255 nm and by molecular mass determinations with electrospray mass spectrometry on a peptic peptide from H. pomatia d and on peptides obtained by proteolysis of carboxymethylated H. pomatia d with trypsin and pronase. Upon sequence analysis the cysteine-histidine thioether bridge was cleaved into didehydroalanine (polymers) and 2-thiolhistidine. A peptide with a cysteine-histidine thioether bridge was isolated from pepsinolysates of functional units c, e, f, g and h of Sepia officinalis and unit g of Octopus vulgaris, obtained from haemocyanin of the cephalopods S. officinalis and O. vulgaris. A cysteine-histidine thioether bridge, which was reported previously for tyrosinase of Neurospora crassa, thus seems to be a general feature for functional units of molluscan haemocyanins.

Further approaches to the quaternary structure of octopus hemocyanin: a model based on immunoelectron microscopy and image processing.

The direction of the polypeptide chains and the location of the functional units in Octopus vulgaris hemocyanin were studied by various methods. Monoclonal antibodies specific for the Ovc (clone Ov409) and Ovg (clone Ov315) functional units produced immunocomplex strings which were examined in the electron microscope. In both cases the immunocomplexes contained more than two hemocyanin molecules in their side view, demonstrating that in the whole hemocyanin neighboring polypeptide chains run in antiparallel directions. The interhemocyanin distances in the immunocomplexes also indicated that Ovg is located inside the cylinder, while Ovc is located in the external layers of functional units. In addition, the fact that the binding point of the Fab arm to the hemocyanin molecule was occasionally visible confirmed the external location of functional unit Ovc. Image processing of the whole hemocyanin cross-linked with dimethyl suberimidate showed that the end-on view is not a perfect cylinder but a regular pentahedron and that the five-arch collar is probably composed of five pairs of functional unit Ovg located inside the cylinder. The accessibility of cross-linked hemocyanin to functional unit-specific polyclonal antibodies, studied in immunoelectrophoresis, showed that Ovb and Ove are highly accessible, while Ovd, Ovf, and Ovg are not. The low accessibility of Ovd may be at least partially explained by its high sugar content which could hamper the accessibility of the antibody to the antigen.

Human urinary protein 1: evidence for identity with the Clara cell protein and occurrence in respiratory tract and urogenital secretions.

Protein 1 (P1), a low mol mass urinary protein of unknown function, has been purified, sequenced and quantified in human biological fluids. The molecular size, subunit composition and partial amino acid sequence of P1 are similar to those of the 10 kDa Clara cell protein (CC10), a lung secretory protein. P1 is found in high concentrations in sputum, bronchoalveolar lavages, urine and semen of healthy individuals and in urine of some pregnant women. Contrary to what is claimed, P1 or CC10 is not a specific and unique product of the lung, but like its homologue in rabbits (uteroglobulin) it is also present in urogenital secretions. P1 or CC10 may act as a natural immunosuppressor protecting the respiratory and urogenital tracts from unwanted inflammatory reactions.

Polar zipper sequence in the high-affinity hemoglobin of Ascaris suum: amino acid sequence and structural interpretation.

The extracellular hemoglobin of Ascaris has an extremely high oxygen affinity (P50 = 0.004 mmHg). It consists of eight identical subunits of molecular weight 40,600. Their sequence, determined by protein chemistry, shows two tandemly linked globin-like sequences and an 18-residue C-terminal extension. Two N-linked glycosylation sites contain equal ratios of mannose/glucosamine/fucose of 3:2:1. Electron micrographs suggest that the eight subunits form a polyhedron of point symmetry D4, or 42. The C-terminal extension contains a repeat of the sequence Glu-Glu-His-Lys, which would form a pattern of alternate glutamate and histidine side chains on one side and of glutamate and lysine side chains on the other side of a beta strand. We propose that this represents a polar zipper sequence and that the C-terminal extensions are joined in an eight-stranded beta barrel at the center of the molecule, with histidine and glutamate side chains inside and lysine and glutamate side chains outside the barrel compensating each other's charges. The amino acid sequence of Ascaris hemoglobin fails to explain its high oxygen affinity.

The reaction of nitrogen monoxide with the haemocyanins of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The rate of the reaction of Astacus leptodactylus methaemocyanin with NO follows the Henderson-Hasselbalch equation with a pKa of 5.85, suggesting that one imidazole ligand of Cu was exchanged for NO. The reaction is blocked by F- as a bridging ligand. The same imidazole residue might be responsible for the decomposition of nitrosylhaemocyanin, [Cu1NO+CuII], with an unlocated binding site for NO, into methaemocyanin and NO, as the rate increase with pH. NO could react preferentially with CuA of Helix pomatia methaemocyanin, CuA'IICuBII, as it possibly has only two histidine ligands instead of the three of CuA in Astacus haemocyanin. This difference might explain the higher formation rate and the much greater stability of Helix nitrosylhaemocyanin. The fast reaction is governed by a pKa of 6.80, probably of a bridging mu-aquo ligand. With F- or a mu-hydroxo bridging ligand a low reaction rate was still observed, in contrast with Astacus methaemocyanin. Helix nitrosylhaemocyanin was transformed by N3- into methaemocyanin with the liberation of N2 and N2O. This methaemocyanin could almost quantitatively be regenerated with H2O2. Helix nitrosylhaemocyanin was only partially regenerated by a direct treatment with H2O2 and almost quantitatively by HONH2 in a similar fairly fast reaction, followed by a much slower one.

Complete amino-acid sequence of a functional unit from a molluscan hemocyanin (Helix pomatia).

From the beta c-hemocyanin (beta c-Hc) of the vineyard snail, Helix pomatia, the functional unit d (Mr approximately equal to 50,000-55,000) was isolated by limited proteolysis and gel chromatography. A small quantity of functional unit d was obtained intact, but the major part in the form of two peptides (Mr approximately equal to 43,000 and 10,000, respectively) connected by a disulfide bridge. After reduction and carboxymethylation, these were separated from each other and cleaved by conventional methods. The peptides were isolated by gel chromatography and HPLC, and sequenced manually or automatically. The complete sequence of Helix beta c-Hc d comprises 410 residues plus 3 residues at the N-terminus seemingly resulting from incomplete cleavage. There is apparently only one carbohydrate side-chain. Comparison of this gastropodan hemocyanin sequence to the partial sequence of a cephalopodan Hc C-terminal unit revealed sufficient identities to state that the functional units of molluscan hemocyanins have arisen by a series of gene duplications. On the other hand, there is practically no homology with arthropodan hemocyanins except for one section of 42 residues which is clearly homologous. This section corresponds to the "Copper B" site of Panulirus interruptus hemocyanin. It is also found in tyrosinases from Neurospora crassa, Streptomyces glaucescens, and mouse. In the N-terminal half of Helix beta c-Hc d there are other sections clearly homologous to the tyrosinases, but overall homology is limited. The second copper-binding site was not identified but must be completely distinct from the "Copper A" binding site of arthropodan hemocyanins. It is suggested that molluscan and arthropodan hemocyanins have evolved independently from a common ancestral mononuclear copper protein.

Interaction of alpha-lactalbumin with dimyristoylphosphatidylcholine vesicles. III. Influence of the temperature and of the lipid-to-protein molar ratio on the complex formation.

We investigated the interaction between alpha-lactalbumin and sonicated dimyristoylphosphatidylcholine at pH 4 and different temperatures. (1) At 23 degrees C and lipid-to-protein molar ratios below 170, the interaction results in a disruption of the original vesicles to form smaller complex particles. By the sedimentation velocity method we determined for this particle a molar mass of (1.05 +/- 0.16) X 10(6) g X mol-1. The lipid-to-protein molar ratio within the complex particle is 70/1, as earlier estimated. It follows that there are approximately 1200 lipid and 17 alpha-lactalbumin molecules per particle. At molar ratios above 170, alpha-lactalbumin strongly associates with the vesicles. In this case the vesicle entity remains. The ability of alpha-lactalbumin to break up the vesicles at this temperature is determined by the number of protein molecules which are required in the complex particle. (2) By means of fluorescence polarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene and energy transfer of the tryptophan groups of the protein to 1,3-(1,1'-dipyrenyl)propane located in the hydrocarbon region of the vesicles, it is shown that with increasing temperature above 25 degrees C, complexes of decreasing internal lipid-to-protein molar ratio are formed. However, by electron microscopy we show that the overall size of these complexes remains approximately the same, i.e., bars with dimensions 70 X 220 A. A temperature-reversible transformation occurs between these complexes, which cannot be isolated by gel chromatography. In contrast, the complex of molar ratio 70/1 remains stable at lower temperatures.

Stabilization of whole molecules of gastropodan haemocyanins by chlorhexidine.

Chlorhexidine diacetate 0.01% stabilized the whole molecules of Helix pomatia alpha-haemocyanin under dissociating conditions; in 1 M NaCl and in 1.42 M sucrose. Dissociation at low ionic strength (10 mM) of the haemocyanin of Pila leopoldvillensis was likewise prevented by chlorhexidine. After dissociation into half molecules of H. pomatia alpha-haemocyanin in 1.42 M sucrose and of P. leopoldvillensis haemocyanin by lowering the ionic strength, whole molecules were formed on introduction by dialysis of chlorhexidine diacetate to a concentration of 0.01%. This stabilization points to the binding of the two chlorophenyl groups in hydrophobic regions of the protein and an ensuing cross-linking of the half molecules of gastropodan haemocyanins. Chlorguanide, which almost corresponds to one half chlorhexidine, even at a concentration of 0.1%, only slightly stabilized the whole molecules.

Fragmentation of crystalline beta-haemocyanin of Helix pomatia with plasmin and trypsin. Location of the fragments in the polypeptide chain.

The action of plasmin on tenth molecules of beta C-haemocyanin of Helix pomatia at pH 8.2 yielded first a three-domain fragment P3 and a five-domain fragment P1 (present as a dimer at pH 8.2). Fragment P1 was further split by plasmin into a four-domain fragment P2 and a one-domain fragment P4 (also present as a dimer at pH 8.2). Trypsinolysis of P2 yielded T2 and fragment X, which was further split into T1C and T3. Fragments P3 and P4 corresponded respectively to the tryptic fragments T1A and T1B, also by their circular dichroic spectra. The determination of the N-terminal groups and the order of splitting allowed the location of the fragments in the polypeptide chain: P3 (a--c), P2 (d--g), P4 (h); T1A (a--c), T3 (d), T1C (e--f), T2 (g), T1B (h).

Iso-cytochrome c species from baker's yeast. Analysis of their circular-dichroism spectra.

The circular-dichroism spectra of baker's-yeast iso-1- (methylated and unmethylated forms) and iso-2-cytochrome c species were examined between 200 and 600nm. In the visible region the yeast haemoproteins have characteristics nearly indistinguishable from those of horse heart cytochrome c. From the spectra in the u.v. region the latter appears, however, to be more helical. It is proposed that the likely element of non-helical structure in iso-1-cytochrome c is residues 62-70.

Limited trypsinolysis of beta-haemocyanin of Helix pomatia. Characterization of the fragments and heterogeneity of the copper groups by circular dichroism.

A limited trypsinolysis of the tenths of beta-haemocyanin of Helix pomatia was performed at pH 8.2. The absorbance at 346 nm remained constant, indicating a preservation of the oxygen-binding sites. The five tryptic fragments were separated by chromatography on Sephadex G-100 and on DEAE-cellulose. They contained 2 Cu per 50000 daltons and showed different mobilities in agar electrophoresis. The molecular weights indicated that one fragment was constituted of three functional domains of about 50000 daltons, that two fragments were constituted of two domains, and two others of one domain. Twentieths of beta-haemocyanin seemed thus to be made up of 9 domains. The circular dichroic spectra of the fragments indicated the presence of two classes of copper groups according to their positive maximum at 455 or at 500 nm. The circular dichroic spectra also showed that no fragment could have originated from a larger one, confirming the presence of nine domains in the twentieths.