Crystallization and preliminary crystallographic studies of calgranulin C, an S100-like calcium-binding protein from pig granulocytes.

Calgranulin C (CAGC) from pig granulocytes has been crystallized and X-ray diffraction data have been collected to 2.6 A resolution. The crystals belong to the trigonal system, space group P3(1)21 or P3(2)21, cell parameters a = b = 54.35 (2), c = 141.32 (5) A and probably contain two molecules in the asymmetric unit. CAGC is amongst the first reported typical S100-1ike calcium-binding protein to be crystallized and studied by X-ray crystallography.

Purification and structural characterization of a fatty acid-binding protein from the liver of the catfish Rhamdia sapo.

We report here the isolation of a fatty acid-binding protein (FABP) from the liver of the catfish Rhamdia sapo. The purification procedure involves gel filtration, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The purified protein is basic (pI > 8.7) and migrates on sodium dodecyl sulfate-gel electrophoresis as a single entity of about 15 kDa. Its amino acid composition resembles those of FABPs isolated from other animals. Unlike mammalian liver FABPs, catfish liver FABP contains at least one tryptophan residue per molecule. No significant cross-reactivity was observed between the purified protein and polyclonal antibodies against either rat liver FABP or rat heart FABP. Amino acid sequencing of peptides obtained by digestion with Lys-C revealed that the catfish protein is structurally more similar to chicken liver FABP (69% identity in a 67-residue overlap) than to human liver FABPs (36%), nurse shark (Ginglymostoma cirratum) liver FABP (30%) and human heart FABP (31%). Taken together, these results suggest that catfish liver FABP is far more closely related to chicken liver FABP than to the FABPs isolated from the liver of mammals or elasmobranchs.

Purification, characterization, and partial amino acid sequencing of an amphibian liver fatty acid binding protein.

The fatty acid binding protein (FABP) from toad liver cytosol was purified to homogeneity by a procedure involving gel filtration and anion exchange chromatography. The protein presented a molecular mass of 13 987 +/- 2 daltons determined by electrospray mass spectrometry. Native isoelectric focusing of the purified liver FABP revealed a single pI 6.8 band. On the other hand, the toad heart FABP showed a different mobility than that of toad liver FABP by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Moreover, toad liver FABP cross-reacted with antisera to mammalian liver FABP but not with antisera to heart FABP. The difference between toad liver and heart FABPs was further confirmed by partial amino acid sequencing. As the N-terminus of toad liver FABP was blocked, the protein was chemically and enzymatically cleaved and the resulting peptides were subjected to automated Edman degradation. Partial amino acid sequencing showed that the toad liver FABP is related to that of mammalian liver and is clearly different from the amphibian heart FABP as well as from the amphibian intestine FABP.

Complex assembly of calgranulins A and B, two S100-like calcium-binding proteins from pig granulocytes.

Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calcium-binding proteins that in human, bovine and mouse granulocytes are associated into a heterocomplex. We have previously identified in pig granulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated with CAGC, we herein investigate its possible association with other proteins. CAGA was purified from pig granulocytes by gel filtration followed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass spectrometry, chemical cross-linking and hydrophobic interaction chromatography. The CAGA-associated protein was further characterized by amino acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protein; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by internal amino acid sequencing as the porcine homologue of CAGB. The stoichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. Unlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column in a calcium-dependent manner. Our results suggest that pig granulocytes contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heterodimer. It is proposed that CAGB/CAGB and the CAGA homodimer may play different roles in vivo.

Molecular evolution of the multigene family of intracellular lipid-binding proteins.

The intracellular lipid-binding proteins are a group of homologous proteins which bind and facilitate the transport of fatty acids, bile acids and retinoids. The evolutionary relationship of 51 family members from vertebrates and invertebrates was analyzed. The inferred phylogeny implies the occurrence of at least 14 gene duplications and contains five regions where the branching order is statistically non-significant--this uncertainty explaining most inconsistencies between previous phylogenetic analyses. The phylogeny also suggests that the intestinal fatty acid-binding protein and the liver fatty acid-binding protein/ileal lipid-binding protein subfamilies diverged from the other subfamilies before the vertebrate-invertebrate split. Finally, results presented herein indicate that the putative fatty acid-binding domain of NMDA receptor 1 is unlikely to be a member of this family.

Primary structure and binding properties of calgranulin C, a novel S100-like calcium-binding protein from pig granulocytes.

In this paper we report the biochemical characterization of calgranulin C, a new member of the S100 protein family. The protein is highly abundant in the cytosol of pig granulocytes, with relatively small amounts in lymphocytes. A simple protocol for the rapid purification of calgranulin C is described. The purified protein migrates as a single entity on SDS-polyacrylamide gel electrophoresis while it has two isoforms focusing at pH 5.8 and 5.5. Gel filtration and cross-linking experiments indicate that calgranulin C is capable of dimerization. The complete amino acid sequence was determined by Edman degradation of peptides generated by trypsin and V8 protease digestion. Calgranulin C consists of 91 residues and has a calculated molecular mass of 10,614 daltons. This value is virtually identical to that obtained by electrospray mass spectrometry. Sequence analysis predicts two EF-hand calcium-binding motifs, the first having an extended loop that is distinctive of the S100 protein family. The metal-binding properties were studied by means of a direct 45Ca(2+)-binding assay and by tyrosine fluorescence titration. Calgranulin C binds not only calcium but also zinc ions. A single high affinity Zn(2+)-binding site per monomer was evidenced by fluorimetric titration. Zinc binding to calgranulin C induces a remarkable increase in the protein affinity for calcium; in the absence of zinc, the protein binds 1 Ca2+/monomer with a binding constant of about 2 x 10(4) M-1, whereas the Zn(2+)-loaded form binds 2 Ca2+/monomer with Ka values of approximately 3 x 10(7) and 6 x 10(4) M-1. Circular dichroism analysis showed that the binding of calcium to calgranulin C induces a 15% decrease in the apparent alpha-helix content. This result and the calcium-dependent binding of the protein to a phenyl-Superose column strongly suggest that calgranulin C undergoes a gross conformational change upon calcium binding, thus supporting the idea that this protein may be involved in Ca(2+)-dependent signal transduction events.

Purification, characterization and partial sequencing of the heart fatty acid-binding protein from Bufo arenarum.

A 15.7 kDa fatty acid-binding protein from toad heart was purified by gel-filtration chromatography on Sephadex G-75 followed by anion-exchange chromatography on a Mono-Q column. Purity was confirmed by gel electrophoresis and isoelectric focusing. Molecular mass, isoelectric point, amino acid composition and partial internal sequence showed that the protein is related to mammalian heart fatty acid-binding proteins.

Isolation and N-terminal sequence of two low molecular weight calcium-binding proteins from pig granulocytes.

1. Two small, abundant calcium-binding proteins were isolated from pig granulocytes. They were named p7A and p7B. Relative molecular masses were approx. 32,000 for p7A and 13,000 for p7B, when obtained by Sephadex G-75 gel filtration, while it was 7000 for both proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2. N-terminal sequence analysis suggests that p7A is homologous to human and mouse MRP-8 and that p7B may be related to human and mouse MRP-14, though some properties of the latter--such as mobility on SDS-PAGE--were found to be different. In addition, p7A and p7B could be resolved under native conditions, contrasting with the fact that human and mouse MRP-8/MRP-14 form noncovalent complexes.