The kinetic mechanism of serpin-proteinase complex formation. An intermediate between the michaelis complex and the inhibited complex

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.     

A New Current?Voltage Relation for Duct Precipitators Valid for Low and High Current Densities

A closed-form analytic current-voltage formula for duct electrostatic precipitators is presented. A short discussion of previous theoretical and numerical solutions is given, followed by an explanation of the theoretical formula derived here. A comparison with experimental data is then given, showing that the present formula is accurate over a wide range of conditions, including wide plate spacing.     

A photolabile oligodeoxyribonucleotide probe of the decoding site in the small subunit of the Escherichia coli ribosome: identification of neighboring ribosomal components

In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 1397-1405 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 1397-1405 lie within a single-stranded sequence that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S subunits, the photolabile oligodeoxyribonucleotide probe site-specifically incorporates into proteins S1, S7, S18, and S21 (identified by SDS-PAGE, RP-HPLC, and antibody affinity chromatography) and into three separate 16S rRNA regions, specifically, nucleotides A-1396, G-1405-A-1408, and A-1492 and A-1493. These results provide clear evidence that G-1405 in 16S rRNA is within 24 A (the distance between G-1405 and the photogenerated nitrene) of proteins S1, S7, S18, and S21 and each of the other nucleotides mentioned above, consistent with other studies of 30S internal structure. Although the probe binds to inactive 30S subunits about as well as to activated 30S subunits, photolysis of the inactive 30S.probe complex leads to a very different pattern of protein labeling, providing strong evidence, at the protein level, that the inactive to activated transition is accompanied by conformational change in the 1400 region of 16S rRNA.