The isolation of parvalbumin isoforms from the tail muscle of the American alligator (Alligator mississipiensis).
Multiple parvalbumin isoforms have been detected in the tail (skeletal) muscle of the American alligator (Alligator mississipiensis). One of these isoforms (APV-1) has been highly purified and partially characterized. Protein purification involved mainly gel filtration and anion exchange chromatography, and characterization included gel electrophoresis, amino acid composition analysis, metal ion analysis, MALDI-TOF and ESI mass spectrometry, ultraviolet and fluorescence spectroscopy, and one- and two-dimensional 500 MHz proton NMR spectroscopy. The alligator isoforms are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins. In fact, the one highly purified isoform that forms the basis of this study has only phenyl-alanine as an aromatic residue. Ion exchange chromatography further indicates that this isoform has a relatively high isoelectric point (pl approximately 5.0), indicating that it is an alpha-lineage parvalbumin. This alligator parvalbumin isoform is unusual in that it has an atypically high Ca2+ content (almost 3.0 mole of Ca2+ per mole of protein) following purification, a fact supported by terbium fluorescence titration experiments. Preliminary comparative analysis of the highly purified alligator parvalbumin isoform (in the Ca2-loaded state) by two-dimensional 1H-NMR (2D 1H TOCSY and 2D 1H NOESY) indicates that there is considerable similarity in structure between the alligator protein and a homologous protein obtained from the silver hake (a saltwater fish species).
Laney
E L
EL
Gustaf H. Carlson School of Chemistry, Clark University, Worcester, MA 01610, USA.
Shabanowitz
J
J
King
G
G
Hunt
D F
DF
Nelson
D J
DJ
D000489:Alligators and Crocodiles / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000737:chemistry; D010320:Parvalbumins / Q000737:chemistry / Q000302:isolation & purification*; D010649:Phenylalanine / Q000737:chemistry; D013057:Spectrum Analysis
D000596:Amino Acids; D010320:Parvalbumins; D010649:Phenylalanine; D002118:Calcium