Panulirus interruptus hemocyanin. The elucidation of the complete amino acid sequence of subunit a

Abstract

As a final step in the elucidation of the primary structure of subunit a of Panulirus interruptus hemocyanin (657 residues, M , 75696 excluding two copper ions and carbohydrate), the amino acid sequence of the largest fragment obtained by limited trypsinolysis was determined. The elucidation of the sequence of residues 176 -657, comprising domains two and three, was mainly based on two digests, with CNBr and trypsin, respectively, from both of which a complete set of peptides was obtained. Additional sequence information was obtained from a digest with Staphylococcus aureus V8 protease and from one fragment obtained by cleaving subunit a with hydroxylamine. A block during Edman degradations indicated an Asn-Gly sequence at positions 597 -598, although only aspartic acid was identified at position 597.Hemocyanins are large multi-subunit copper-containing oxygen carriers occurring freely dissolved in the hemolymph of many molluscs and arthropods. Arthropod hemocyanins are composed of hexamers or multihexamers with subunits of about 75000 daltons, each of which contains one binuclear copper site [l, 21. Like in all arthropods [l, 21, hemocyanin of the spiny lobster Panulirus interruptus displays subunit heterogeneity [3]. There are at least three subunit types, named a, b and c, which were formerly called '94K', '90K' and '80K', respectively, since their apparent molecular masses on SDS/ polyacrylamide gels were 94, 90 and 80 kDa [3, 41. These subunits have been characterized by examination of their electrophoretic, chromatographic and immunological properties [3, 51. Subunits a and b, accounting for 80-9o0/0 of the monomers upon dissociation of the hexamers, are very similar, but subunit c is quite different [3, 6 -81. Both X-ray diffraction studies and amino acid sequence studies are being performed. The three-dimensional structure has been determined at a resolution of 0.32 nm with crystals containing a mixture of subunits a and b in roughly equal amounts [4, 93. Each polypeptide chain is folded into three distinct domains.Primary-structure investigations were started with subunit a, which gave rise to three fragments upon limited trypsinolysis : an N-terminal 18-kDa fragment, a C-terminal 55-kDa fragment (formerly called 71-kDa fragment after its apparent molecular mass on SDS-PAGE) and a glycopeptide located in between [lo]. The 18-kDa fragment and the glycopeptide together form the first domain while the 55-kDa fragment accounts for domains two and three. The second domain contains the active site, with two copper atoms. Each copper atom is ligated by three histidines, two of which are provided by a -His-Xaa-Xaa-Xaa-His-sequence located in a helix, the third coming from another helix.The sequences of the first 230 residues, comprising the 18-kDa fragment, the glycopeptide, and of 55 N-terminal residues of the 55-kDa fragment have already been reported [lo-121. Partial or complete sequences of other parts have been published as well, but without the evidence [4, 6,8,13, 141. In this paper, the complete eviden...

Panulirus interruptus hemocyanin

“…1 weak spot of Asn at position 405 observed only once during Edman degradation with DABITC and the presence of multiple peaks during reversed-phase HPLC of CNBr peptides containing this sequence, the latter of which showed different charges [9]. In the present study, direct evidence for the presence of asparagine at position 405 was obtained in neither subunit a nor subunit b (peptides CB12H3).…”

“…Most peptides were sequenced manually as described by Chang [12] with slight modifications [9]. In most cases, isoleucine, leucine and the C-terminal residues of peptides were determined by dansylation.…”

“…Subunits a and b, accounting for 80 -90% of the monomers upon dissociation of the hexamers, are very similar, but subunit c is quite different [3, 6-81. The complete amino acid sequence of subunit a has been published recently [9]. The three-dimensional structure has been determined at 0.32-nm resolution [4, 101.…”

“…Subunits a and b, accounting for 80 -90% of the monomers upon dissociation of the hexamers, are very similar, but subunit c is quite different [3,. The complete amino acid sequence of subunit a has been published recently [9]. The three-dimensional structure has been determined at 0.32-nm resolution [4, 101. Since the latter study was performed with crystals containing a mixture of subunits a and b in roughly equal amounts [4, lo], knowledge of the amino acid sequence of subunit b is essential for a reliable interpretation of the electron-density map.…”

Developmental Changes in Hemocyanin Expression in the Dungeness Crab, Cancer magister

“…Because the Cu A -binding site in arthropod Hc domain 2 is highly conserved in all crustacean and chelicerate Hc subunits thus far sequenced (19), we predicted that it would be a conserved feature in all six C. magister Hc subunits as well. Accordingly, primer Cu A I (5Ј-GAA-CTT-TTT-TTT-TGG-GTT-CAT-CAT-CAA-CTT-AC-3Ј), a 32-bp degenerate oligonucleotide, was designed based on the amino acid sequence NH 2 -ELFFWVHHQLT-COOH within the Cu A site of Hc subunit a of the spiny lobster Panulirus interruptus (20). Reverse translation of part of the unique N-terminal amino acid sequence of Hc subunit 6 (Fig.…”