Homoserine kinase from Escherichia coli K-12: properties, inhibition by L-threonine, and regulation of biosynthesis

We have partially purified homoserine kinase from a genetically derepressed strain of Escherichia coli K-12. The optimum pH of the enzyme-substrate reaction was 7.8 and the K(m) values for l-homoserine and adenosine 5'-triphosphate were both 3 x 10(-4) M. K(+) (or NH(4) (+)) as well as Mg(2+) were required for its activity. The sedimentation coefficient determined by ultracentrifugation in a sucrose density gradient was 5.0 +/- 0.25S. l-Homoserine was an excellent protector against heat inactivation of homoserine kinase. l-Threonine was a competitive inhibitor of homoserine kinase, suggesting that end-product inhibition of this enzyme plays a role in vivo in the overall regulation of threonine biosynthesis. The specific activity of aspartokinase I-homoserine dehydrogenase I and of homoserine kinase showed a strong positive correlation in extracts from strains under widely varying conditions of genetic or physiological derepression; it was concluded that these two enzymes are coordinately regulated in E. coli K-12.     

The immunofluorescent demonstration of cathepsin B1 in tissue sections

Summary A direct immunohistochemical method of high specificity is presented for the demonstration of sites of cathepsin B1. Antisera were raised in rabbits against pure enzyme preparations. The specific antibodies were subsequently isolated and labelled according to methods previously published. Sections from tissues specially rich in this enzyme like liver, kidney, intestinal epithelia and the active macrophages of talcum granulomas were studied. The results suggest a localization of this enzyme mainly to lysosomal organelles and phagocytic vacuoles, which are known from chemical data to contain this enzyme. The prerequisite for a positive tissue reaction is that the enzyme must be present in a form available for antigen-antibody interaction.