Studies on the membrane-associated proteolytic activities of Escherichia coli

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1986

OSTI ID:5281313

Palmer, S M

The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl approx. = 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH approx. = 8.0. Incubation of whole membrane preparations with (/sup 3/H) DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind (/sup 125/I) ampicillin.

• Research Organization: Rutgers--the State Univ., New Brunswick, NJ (USA)
• OSTI ID: 5281313
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BETA DECAY RADIOISOTOPES
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ELECTRON CAPTURE RADIOISOTOPES
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
ESCHERICHIA COLI
FRACTIONATION
HYDROLASES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MEMBRANES
MICROORGANISMS
NUCLEI
ODD-EVEN NUCLEI
PEPTIDE HYDROLASES
PROTEOLYSIS
RADIOISOTOPES
SEPARATION PROCESSES
TRACER TECHNIQUES
TRITIUM COMPOUNDS