Binding of oxygen and carbon monoxide to the hemocyanin from the spiny lobster.

A high precision, two-dimensional study of oxygen and carbon monoxide binding to Panulirus interruptus hemocyanin has been carried out. Global data analysis of three types of experiments, probing the molecule in its various states of CO and O2 ligation, revealed the entire hexamer to be the basic allosteric unit involved in a two-state mechanism. The co-operativity and linkage of the two ligands are presented in terms of derivative Hill plot surfaces extended along co-ordinates of CO and O2 activities giving a detailed and comprehensive view of the binding behavior. Among the findings is an apparent high co-operativity of carbon monoxide binding at high oxygen activity. The results are discussed in view of a general mechanism for co-operative behavior found in larger hemocyanin aggregates concerning "nested" allosteric interactions.

Panulirus interruptus hemocyanin. The amino acid sequence of subunit b and anomalous behaviour of subunits a and b on polyacrylamide gel electrophoresis in the presence of SDS.

The primary structure of subunit b of Panulirus interruptus hemocyanin has been derived from two digests (trypsin and CNBr) and, in some cases, with aid from the similarity with the sequence of subunit a. Differences between the amidation states of Asx and Glx residues in subunit b relative to a were investigated more thoroughly. When compared to the sequence of subunit a, 18 differences (2.7%) were found and certain heterogeneities, indicating the presence of a minor subunit b', were observed. Several differences in properties between subunits a and b, including their anomalous behaviour on SDS/polyacrylamide gel electrophoresis, could be explained by amino acid replacements.

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

As a final step in the elucidation of the primary structure of subunit a of Panulirus interruptus hemocyanin (657 residues, Mr 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.

Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli.

Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.

The amino-acid sequence of the three smallest CNBr peptides from p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

After CNBr cleavage of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, five peptides and free homoserine were isolated (see preceding paper in this journal). The amino acid sequences of the three smallest peptides, viz. CB3, CB4 and CB5, were determined by automated Edman degradation and analysis of enzymatic subdigests. These peptides form a continuous stretch of 110 residues from the N terminus: (Formula: See Text).