ABSTRACT
The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.
Subject(s)
Recombinant Proteins/analysis , Amino Acid Sequence , Chemical Phenomena , Chemistry, Physical , Chromatography, High Pressure Liquid , Cyanogen Bromide , Humans , Hydrolysis , Mass Spectrometry , Molecular Sequence Data , Molecular Weight , Sequence Analysis , Serine Endopeptidases , Serum Albumin/analysis , Trypsin , von Willebrand Factor/analysisABSTRACT
In the present study, highly specific radioimmunoassays were developed and used to measure neurokinin B, neurokinin A and substance P in the rat spinal cord and various peripheral tissues. The results are as follows. (1) Neurokinin B and neurokinin A were distributed all along the rostrocaudal axis of the spinal cord, as is substance P, and were more concentrated in the dorsal than in the ventral region. (2) Substance P was more abundant in the central and peripheral nervous tissues than neurokinin A, while in certain peripheral organs, neurokinin A was more abundant than substance P. In the spinal cord, neurokinin B concentrations were lower than those of the other two tachykinins. (3) In contrast to neurokinin A and substance P, neurokinin B was not detected in any of the peripheral tissues examined. (4) Capsaicin treatment reduced by half neurokinin A and substance P concentrations in the dorsal region of the spinal cord, the dorsal root ganglia and the sciatic nerve, but was without effect on neurokinin B concentrations in the spinal cord. Neurokinin A, like substance P, may therefore have an important function in the transmission of sensory information, particularly in nociceptive transmission from the periphery to the spinal cord and in peripheral neurogenic inflammation. In contrast, since neurokinin B was not found in the sensory neurons, it is not likely to have these functions, but may perhaps control them.
Subject(s)
Capsaicin/pharmacology , Ganglia, Spinal/metabolism , Neurokinin A/metabolism , Neurokinin B/metabolism , Sciatic Nerve/metabolism , Spinal Cord/metabolism , Substance P/metabolism , Animals , Animals, Newborn , Ganglia, Spinal/drug effects , Guinea Pigs , Male , Neurokinin A/analysis , Neurokinin B/analysis , Organ Specificity , Radioimmunoassay , Rats , Rats, Inbred Strains , Sciatic Nerve/drug effects , Spinal Cord/drug effects , Substance P/analysisABSTRACT
Two hours after the injection of (3-[125I]iodotyrosyl3) neurotensin into the striatum, a labeling was observed in the ipsilateral substantia nigra. In the present study, we demonstrated by HPLC that this radioactivity corresponded to intact neurotensin and to degradation products of this peptide. This finding provides the first clearcut evidence that a neuropeptide can be internalized and retrogradely transported in brain neurons. Therefore, the fact that intact neurotensin can be seen to exist over a long period of time in the cell body suggests that the retrograde transport process could perhaps be involved in the long-term effects of neuropeptides.
