Ben Neuteboom

STRUCTURE OF ARTHROPOD HEMOCYANIN

Hemocyanins are large multi‐subunit copper proteins that transport oxygen in many arthropods and molluscs. The amino acid sequence of subunit a of Panulirus interruptus hemocyanin (657 residues) has been completed and fitted to the electron‐density map (3.2 Å resolution). Comparison of amino acid sequence data for several different hemocyanin subunits of arthropod species indicated that the general features of the polypeptide architecture as found in spiny lobster hemocyanin occur in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, 540–600 million years ago.

The Unusual Amino Acid Triplet Asn–Ile–Cys Is a Glycosylation Consensus Site in Human α-Lactalbumin

Human α-lactalbumin has not been described as a glycoprotein, despite the fact that several α-lactalbumins of both ruminant and nonruminant species are known to be glycosylated. In all these species the glycosylation site is the 45Asn in the usual triplet 45Asn–Gly/Gln–47Ser. We have found that human α-lactalbumin is glycosylated and the glycosylation site has been determined by protein sequencing and mass spectrometry. We report an unusual glycosylation site at 71Asn in the triplet 71Asn–Ile–73Cys, which is conserved in all known α-lactalbumins except red-necked wallaby. That a relatively small proportion of the protien is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of α-lactalbumin.

The relationship between N-terminal sequences and immunological characterization of crustacean hemocyanins

1. N-terminal amino acid sequences of seven crustacean hemocyanin subunits were determined and compared together with a number of known N-terminal sequences. 2. Within crustacean infraorders, relationships of subunits were established. Relationships based on N-terminal sequences were compared with a classification based on immunological characterization.

Origin of the duplicated ribonuclease gene in guinea-pig: Comparison of the amino acid sequences with those of two close relatives: Capybara and cuis ribonuclease

SummaryThe amino acid sequences of the pancreatic ribonuclease from capybara (Hydrochoerus hydrochaeris) and cuis (Galea musteloides) were determined. Both species belong to the same superfamily of the hystricomorph rodents as the guinea-pig. In guineapig pancreas two ribonucleases are present as a result of a rencent gene duplication, but in capybara and cuis pancreas only one single ribonuclease has been found. A most parsimonious tree of ribonucleases indicates that the gene duplication leading to both guinea-pig ribonucleases occurred before the divergence of guineapig from capybara and cuis. This would mean that changes in expression of the ribonuclease genes have occurred in these taxa.Cuis and capybara ribonuclease have no Asn-X-Ser/Thr sequences and are carbohydrate-free proteins. Capybara ribonculease has leucine at position 114, a position occupied by proline in thecis-configuration in bovine pancreatic ribonuclease.

Primary structure of hemocyanin from Palinurus vulgaris

The primary structure of hemocyanin from the spiny lobster Palinurus vulgaris was determined using a mixture of at least four slightly different subunits. Heterogeneities were observed in 32 (5%) of the positions. The amino acid sequence differs at about 20% of the positions from that of subunit a of Panulirus interruptus hemocyanin.

Sulfhydryl groups and disulfide bridges in subunit c of Panulirus interruptus hemocyanin

Subunit c of Panulirus interruptus hemocyanin contains, after reduction, three cysteine residues. Determination of sulfhydryls in the native subunit revealed one free SH group. The cysteine-containing peptides were obtained from a pepsin digest. A disulfide bridge was established between Cys 3 and Cys 557 , while Cys 99 bears a free sulfhydryl group. These results are in agreement with expectations based on the three-dimensional structure of subunit a . The position of the disulfide bridge in subunit c is unique among arthropodan hemocyanins, and is possible responsible for the absence of cooperativity in subunit c homo-hexamers.

Isolation of a new ligand-carrying casein fragment from bovine mammary gland microsomes.

Whilst looking for components involved in retinol metabolism in secreting mammary gland cells, a 12 kDa Protein was isolated. This protein had bound a ligand with characteristics of retinol. N‐Terminal sequencing and amino acid analysis showed that this protein is highly homologous with an α‐sl‐casein fragment. No ligand was found for β‐lactoglobulin, previously thought to be involved in retinol metabolism.

Subunits a, b and c of Panulirus Interruptus Hemocyanin and Evolution of Arthropod Hemocyanins

Hemocyanins are large, non-haem, oxygen-carrying proteins occurring freely dissolved in the hemolymph of molluscs and arthropods.