Purification of hog renin by affinity chromatography using the synthetic competitive inhibitor (D-Leu6)octapeptide.

The renin substrate analog His-Pro-Phe-His-Leu-D-Leu-Val-Tyr ([D-Leu6]-octapeptide) acts as a potent inhibitor of renin because of the D-amino acid substitution at the cleavage site. This inhibitor was coupled to CNBr-activated Sepharose 4B to yield a support for affinity chromatography. Hog renin with a specific activity of 1.2 Goldblatt units/mg was in one step purified 195-fold to a final specific activity of 234 Goldblatt units/mg. Application of a pH gradient from 5.0 to 7.5 to the support was found to be the most successful elution program, probably because the [D-Leu6]-octapeptide is not an inhibitor for renin at neutral pH.     

Biological antioxidants

The mechanism of lipid peroxidation and the ways in which the rate of this reaction can be reduced by small quantities of certain specific chemicals, called antioxidants, are described. The types and roles of the different antioxidants found in living systems are considered. Vitamin E (alpha-tocopherol) has long been recognized as an important lipid-soluble, chain-breaking antioxidant. It has an unexpectedly high reactivity towards peroxyl radicals, which can be understood only after detailed consideration of its structure. It is the major antioxidant of its class in human blood and its effectiveness in plasma is greatly improved by a synergistic interaction with water-soluble reducing agents such as ascorbic acid. Experiments designed to locate vitamin E within phospholipid bilayers and to discover the origin of the different biopotencies of stereoisomers of alpha-tocopherol are also described.     

Mechanistic Study of Classical Translocation-Dead SpoIIIE36 Reveals the Functional Importance of the Hinge within the SpoIIIE Motor

SpoIIIE/FtsK ATPases are central players in bacterial chromosome segregation. It remains unclear how these DNA translocases harness chemical energy (ATP turnover) to perform mechanical work (DNA movement). Bacillus subtilis sporulation provides a dramatic example of intercompartmental DNA transport, in which SpoIIIE moves 70% of the chromosome across the division plane. To understand the mechanistic requirements for DNA translocation, we investigated the DNA translocation defect of a classical nontranslocating allele, spoIIIE36. We found that the translocation phenotype is caused by a single substitution, a change of valine to methionine at position 429 (V429M), within the motor of SpoIIIE. This substitution is located at the base of a hinge between the RecA-like β domain and the α domain, which is a domain unique to the SpoIIIE/FtsK family and currently has no known function. V429M interferes with both protein-DNA interactions and oligomer assembly. These mechanistic defects disrupt coordination between ATP turnover and DNA interaction, effectively uncoupling ATP hydrolysis from DNA movement. Our data provide the first functional evidence for the importance of the hinge in DNA translocation.