NOVEL 3,4-DIHYDROXYPHENYLALANINE-CONTAINING AND 3,4,5-TRIHYDROXYPHENYLANINE-CONTAINING POLYPEPTIDES FROM THE BLOOD-CELLS OF THE ASCIDIANS ASCIDIA-CERATODES AND MOLGULA-MANHATTENSIS

Acetic acid urea extraction of the blood cells of two ascidian species yielded four novel families of polypeptides (3500-5300 Da) containing 3,4-dihydroxyphenylalanine (DOPA) and 3,4, 5-trihydrogyphenylalanine (TOPA) from the Phlebobranch Ascidia ceratodes and two DOPA proteins ( 9-10 kDa) from the Stolidobranch Molgula manhattensis. 3,4,5-Trihydrox yphenylalanine residues have not been found previously in polypeptides in any biological system, The DOPA content of the M. manhattensis pro teins is the highest yet reported for a naturally occurring DOPA prote in. The successful isolation of proteinaceous components from A. cerat odes blood cells requires the incorporation of high concentrations of complexing agent in the extraction buffers to inactivate vanadium(III) which forms intractable organometallic polymers, The A. ceratodes blo od cell polypeptides are all rich in alanine and TOPA residues, have n eutral to basic pi values, and, while all give single bands on acid ur ea-polyacrylamide electrophoresis, they exhibit extensive microheterog eneity, This is reflected by their large molecular weight distribution as determined by matrix-assisted laser desorption ionization-mass spe ctrometry, the variation in the ratio of their 280-nm absorption to ch romophores of unknown structure in the polypeptide's electronic absorp tion spectra, and the N-terminal sequence analysis. The two M. manhatt ensis proteins consist largely of four amino acids (alanine, valine, p henylalanine, and DOPA) with DOPA accounting for upward of 40 mol%, Bo th give an N-terminus of Ala-Phe-Tyr before resisting further progress by Edman degradation. Proteins and polypeptides from both ascidians a re extremely resistant to proteases, a property which, while hampering characterization by sequence analysis, appears ideally suited to thei r proposed function of forming an impervious hemostat at the site of v ascular injury, The yield of proteins and polypeptides relative to tun ichromes appears to be seasonally dependent, The presence of DOPA and TOPA moieties in the proteins and polypeptides implicates them, as wel l as the tunichromes, as potential metal-sequestering agents. (C) 1995 Academic Press, Inc.