Manfred Liersch

Determination of Specific Growth Stages of Plant Cell Suspension Cultures by Monitoring Conductivity Changes in the Medium

SummaryConductivity changes in the medium of cultured soybean (Glycine max L.) cells were shown to be strictly correlated with nitrate uptake and growth of the cultures. A continuous record of the conductivity was used as a simple and reliable method of determining specific growth stages and concomitant peaks in the activities of nitrate reductase and phenylalanine ammonia-lyase.

Crystallization of human leukocyte elastase with its inhibitor Pro44-eglin c

Human leukocyte elastase has been crystallized in complex with recombinant Pro44-eglin c in the orthorhombic space group P2(1)2(1)2(1). The cell constants are a = 126.1 A, b = 127.8 A, c = 69.4 A, alpha = beta = gamma = 90 degrees. The crystals diffract to at least 2.5 A resolution and are suitable for crystallographic structure analysis.

New Inhibitory Properties of Eglin C after Specific Mutagenesis

Eglin c is a small protein containing 70 amino acids. It shows a high inhibitory activity towards several serine proteinases, like human leucocyte elastase (HLE), α-chymotrypsin, and subtilisin. The protein was first isolated from the leech Hirudo medicinalis (Seemuller et al 1977). The determination of the amino acid sequence revealed that the molecule lacks the sulfur-containing amino acids Cys and Met (Seemuller et al 1980). The absence of disulfide bridges has been the basis for the classification of eglin c belonging to the potato I inhibitor family (Svendsen et al 1982). A gene encoding eglin c has been synthesized and expressed in E.coli, yielding a convenient source of recombinant eglin c (Rink et al 1984). The three-dimensional structure of the molecule, in complex with subtilisin Carlsberg was elucidated by x-ray crystallographic techniques, and refined to 1.2 A resolution (McPhalen et al 1985, Bode et al 1986). Eglin c consists of a hydrophobic core and an active site binding loop fixed on opposite sides. The hydrophobic core contains a twisted four-stranded s-sheet and a short α-helical segment (Fig.1). The loop which harbors the “scissile” peptide bond between the amino acids Leu45 and Asp46, is held in a rigid conformation by strong electrostatic and hydrogen bond interactions of Thr44 and Asp46 with the side-chains of Arg51, Arg53 and C-terminus of Gly70.

A Large Fragment Approach to Gene Synthesis

Abstract The total synthesis of a 232 base-pair coding sequence of the proteinase inhibitor eglin c from only six synthetic fragments is described.