Richard N. Harkins

Characterization of intact and trypsin-digested biosynthetic human growth hormone by high-pressure liquid chromatography

Abstract The structural properties of purified human growth hormone (hGH) produced by Escherichia coli K-12 into which the hGH gene has been inserted have been fully characterized by high-pressure liquid chromatography of native hGH and tryptic digests of hGH. All of the tryptic peptides have been separated by high-pressure liquid chromatography and their sequence determined. Comparison of the primary structure with that of the purified pituitary-derived hGH has established the integrity of the biosynthetic hGH disulfide arrangement and amino acid sequence with the presence of an extra NH 2 -terminal methionine.

Design and operation of a completely automated beckman microsequencer

A unique, efficient, and inexpensive system has been designed and built for the automatic conversion of anilinothiazolinone derivatives extracted from a Beckman spinning-cup sequencer with subsequent on-line high-pressure liquid chromatography separation of the phenylthiohydantoin derivatives. The Auto Converter-Auto Sampler system is controlled by a tape programmer or microprocessor and operates by transfer of the sample from the conversion vial into an HPLC injection loop by nitrogen pressure. Incorporation of a minor programming change on the sequencer allows the introduction of nitrogen vapor into the spinning cup during phenylisothiocyanate coupling. These modifications have resulted in a completely automated subnanomole protein sequencer.

Chemical and Biological Properties of Human Lymphotoxin

The cytotoxicity of lymphocytes toward allogeneic target cells in vitro was first shown by Govaerts (1960). This was later confirmed by Rosenau and Moon (1961). It was demonstrated that lymphocytes, when activated with antigen or mitogen, secrete a soluble cytotoxin which was named lymphotoxin (Granger and Kolb, 1968). Since then a number of established human lymphoid cell lines have also been reported to produce a similar cytotoxic molecule (Amino et al., 1974; Granger et al., 1970; Shacks et al., 1973; Papermaster et al., 1976). The activity of lymphotoxin has been tested against a number of cell lines both of animal and human origin. It has been demonstrated by several workers that lymphotoxin is both cytostatic and cytolytic to target cells (Evans and Heinbaugh, 1981; Rosenau, 1981; Sawada et al., 1976). Most of the biological studies with lymphotoxin have been performed with relatively crude preparations. The isolation of lymphotoxin has been a rather difficult task due to its heterogeneous nature and also because of the small amounts produced by normal lymphocytes and various lymphoid cell lines. We have purified human lymphotoxin from several hundred liters of cell conditioned medium derived from the lymphoblastoid cell line RPMI 1788. Using purified material we have also examined the in vitro effects of lymphotoxin on human tumor and normal cells.