Hemocyanins in spiders, XXIII. Complete amino-acid sequence of subunit a of Eurypelma californicum hemocyanin.

The complete amino-acid sequence of subunit a of the hemocyanin of the tarantula Eurypelma californicum was determined by manual sequencing. By limited chymotrypsinolysis, subunit a is split into two fragments of 25 kDa and 40 kDa, respectively, only one single peptide bond being attacked. The whole chain contains 15 methionine residues, after cyanogen bromide cleavage, 15 peptides were identified indicating that one residue (Met85) was not split by the cyanogen bromide reaction. For subcleavages, trypsin, chymotrypsin, Staphylococcus aureus proteinase, and Astacus fluviatilis proteinase were employed. The total chain length comprises 627 amino-acid residues, carbohydrate side chains were not found.

The functional role of cytochrome b5 reincorporated into hepatic microsomal fractions.

Incorporation of detergent-solubilized cytochrome b5 into phenobarbital-induced rabbit liver microsomal fractions decelerates hexobarbital-dependent reduction of ferric cytochrome P-450; this is accompanied by retardation of NADPH utilization and H2O2 formation in the assay media. Integration of manganese-substituted cytochrome b5 into the microsomal preparations fails to affect these parameters. Analysis of the cytochrome P-450 reduction kinetics in the presence of increasing amounts of cytochrome b5 reveals a gradual augmentation of the amplitude of slow-phase electron transfer at the expense of the relative contribution of the fast phase; finally, a slow, apparently monophasic reaction persists. This defect in enzymatic reduction is not due to detergent effects and also does not seem to reflect cytochrome b5-induced perturbation of anchoring of NADPH-cytochrome c(P-450) reductase to cytochrome P-450. Experiments with the highly purified cytochrome P-450 isozyme LM2, in which amino acid residue(s) close to the heme edge had undergone suicidal inactivation through covalent attachment of chloramphenicol metabolite(s) do not exclude the possibility that cytochrome b5 and reductase might compete for a common electron transmission site on the terminal acceptor. Hence, the inhibitory action of cytochrome b5 on the reduction of ferric cytochrome P-450 is tentatively attributed to partial substitution of the former pigment for reductase in direct transport of the first electron to the monooxygenase.

The structure of arthropod hemocyanins.

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

Hemocyanins in spiders, XX. Sulfhydryl groups and disulfide bridges in subunit d of Eurypelma californicum hemocyanin.

Subunit d of Eurypelma californicum hemocyanin contains after reduction 7 cysteine residues. Using 3,3'-dithiobis(6-nitrobenzoic acid) 3 mol cysteine/mol subunit were determined. The cysteine- and cystine-containing peptides of subunit d were obtained by cyanogen bromide cleavage and subsequent treatment with trypsin. The free cysteines were established at positions 102, 261, and 454 respectively. Cys205-Cys210 and Cys529-Cys579 are connected by disulfide bridges.

Hemocyanins in spiders, XIX. Complete amino-acid sequence of subunit d from Eurypelma californicum hemocyanin, and comparison to chain e.

The complete primary structure of subunit d of the hemocyanin from the tarantula Eurypelma californicum was determined by manual micro sequencing. Subunit d of Mr = 73000 is split about in the middle of the chain during limited trypsinolysis, only one single bond being attacked. The whole chain contains 14 methionine residues and after cyanogen bromide cleavage 15 peptides could be isolated by gel and ion exchange chromatography and high pressure liquid chromatography. The cyanogen bromide peptides and the large (Mr = 34000 and 37000, respectively) fragments resulting from limited trypsinolysis, were further cleaved with trypsin, chymotrypsin, Staphylococcus aureus proteinase, formic acid, and Astacus fluviatilis proteinase, the latter being very useful in obtaining certain overlapping peptides. The total chain length is 627 residues. Carbohydrate side chains were not found. The sequence is discussed with respect to the gross physical properties of the subunit, to homologies with subunit e and the cleavage specifities of the enzymes employed.

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Hemocyanins in spiders, X. Limited proteolysis of chain e of Eurypelma hemocyanin and partial sequence of two large fragments.

The polypeptide chain e of the homocyanin from the spider Eurypelma californicum was isolated by ion exchange chromatography. Incubation of the undenatured protein with chymotrypsin, subtilisin, or trypsin resulted in a small number of large fragments which were easily isolated after denaturation. Of the chymotryptic peptides e-Chn-29 was found to be N-terminal, and e-Chn-42 C-terminal. These peptides were characterized by their N-terminal amino acid sequences. The N-terminal sequence of subunit e shows homologies with other arthropod hemocyanins.

Hemocyanins in spiders, VI[1]. Comparison of the polypeptide chains of Eurypelma californicum hemocyanin.

The subunits of the hemocyanin from the tarantula, Eurypelma californicum, were isolated, following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing two chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is seven. The amino acid compositions of the seven chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident, if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in the case of chains b and e,, while no end group called be detected in any of the other chains by different methods. The C-terminal end group was found to be valine in both chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the two largest peptides of each chain are of molecular weight near 2400. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications, but resides in major differences of the amino acid sequences.

Hemocyanins in spiders, IV[1]. Subunit heterogeneity of Eurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains.

The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.