Hans-Hermann Kiltz

Preparation of a highly purified bovine trypsin for use in protein sequence analysis

Abstract Bovine trypsin is purified by stepwise affinity chromatography on phenylbutylamine Sepharose and benzamidine cellulose. The trypsin is shown to be free of chymotrypsin contaminations as demonstrated by digestion of a small peptide and two proteins, which are very accessible to chymotrypsin. It strongly facilitates sequence analysis of hydrophobic proteins.

High-resolution gel filtration of the ecdysteroid receptor-DNA complex —an alternative to the electrophoretic mobility shift assay

The mobility shift assay is a well established method for proving binding of protein to DNA. However, this method depends on the stability of the protein-DNA complex during the electrophoretic process. Ecdysteroid receptor shows a strong tendency to aggregate under low-salt conditions of electrophoresis to a non DNA-binding form. We have developed a high-resolution gel filtration method which allows the interaction of ecdysteroid receptor with specific DNA sequences to be studied. The method seems to be generally applicable. It does not depend on the availability of a purified protein. Crude preparations could be used to characterize the stoichiometry and the molecular parameters of the complexes formed between DNA and DNA-binding proteins.

The Sites of Interaction of Calmodulin with Phosphofructokinase

Calmodulin is a multifunctional calcium-binding protein found in all eukaryotic cells (for review see 1). It consists of four Ca++-binding helix-loop-helix domains which are homologous to each other and are often referred to as the EF-hand structures (2). Upon binding of calcium ions, calmodulin undergoes conformational changes (1,3–5) allowing it to interact with several target proteins.

Step-wise cross-linking of pig heart lactate dehydrogenase by a heterobifunctional reagent☆

Cross-linking of the essential cysteine-165 of pig heart lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) with lysine-179 was achieved by stepwise reaction with N-(4-azidocarbonyl-3-hydroxy-phenyl)-maleimide. These two residues are connected by kind of a hydrophobic channel which suggests that the essential cysteine is not modified via the active center as previously assumed.