Isolation and characterization of two peptides specific to soybean nodules

Abstract

Two low molecular weight peptides, each with a molecular weight of about 5.000, were isolated from soybean nodules. The levels of these peptides were positively correlated with the level of nitrogenase activity. These peptides are nodule-specific and bind ammonia and ureides. Peptide R is composed of 16.57% glutamic acid, and 15.02% aspartic acid, 13.26% alanine, 11.45% proline, 10.45% serine, and low levels of other common amino acids. Peptide Y contains 54.26% aspartic acid and 22.03% glutamic acid, and low levels of other common amino acids. Observations suggest that each peptide contains a transitions metal such as cobalt, copper, molybdenum, or nickel. Spectroscopic analysis revealed undistinguished spectra. Spectra of each peptide show similarities between them and rule out any possibility of either being a nitrogenase fragment. Two chromophores are common to peptides R and Y; peptide Y contains one additional chromophore. ORD spectra indicate a low amount of ordered structure for both peptides. Incorporation of [^15]N from [^15]N2 into free amino acids and the two peptides was measured using plasma desorption mass spectrometry. A 2.1 atom % [^15]N excess was seen for aspartic acid and a 5.7 atom % [^15]N excess for glutamic acid. A 3.2 atom % [^15]N excess incorporation was found for ureides bound to combined peptide sample. A MW 147 peak remains unidentified. Cyanide reduction, an activity associated with nitrogenase, may be enhanced by peptide Y. both peptides act on cyanide but neither reduce acetylene. The non-specificity of substrates common t nitrogenases is not observed with the peptides. Cyanide reduction by both nitrogenase and a combine peptide sample was monitored by [^13]C-NMR. Three transient peaks were observed which were identified as oxygenated imine compounds. The peptides found are suspected to be involved in the transfer of ammonia from the site of reduction to the site of assimilation, acting as carriers... The appearance and disappearance of NMR signal due to transient compounds produced during cyanide reduction was the first observation of intermediates formed during a reduction catalyzed by nitrogenase. These intermediates are the first direct evidence supporting the hypothesis that reduction of dinitrogen occurs via diimide and hydrazine intermediates.