title: Specific labeling of diaminopimelate: a radioassay for the determination of the peptidoglycan cross-linking index.
abstract: A radioassay for the determination of the peptidoglycan cross-linking index (CLI) was devised. It is based on specific radioactive labeling of diaminopimelic acid (DAP) by diverting [14C]aspartate into the DAP pathway, while inhibiting incorporation of label into other cell wall components. Purified [14C]DAP-labeled cell walls were treated with fluorodinitrobenzene, hydrolyzed, and chromatographed by TLC. The radioactivity in well-separated mono dinitrophenyl-diaminopimelate (DNP-DAP) and DAP spots was counted and the CLI was determined from the ratio of DAP to the total of mono DNP-DAP and DAP counts. The method, suitable for bacteria such as Bacillus subtilis unable to incorporate exogenous DAP, can be applied to other systems. A CLI of 50.8 +/- 1.3% and 55.5 +/- 0.9% was obtained for B. subtilis 168 cells growing exponentially in rich and minimal medium, respectively. Comparison of these to results previously obtained on B. subtilis suggested the existence of a hitherto unreported peptidoglycan endopeptidase activity.
authors.affiliation: Institut de génétique et de biologie microbiennes, Lausanne, Switzerland.
authors.forename: C A
authors.initials: CA
authors.lastname: Roten
authors.forename: M
authors.initials: M
authors.lastname: Pagni
authors.forename: P
authors.initials: P
authors.lastname: Margot
authors.forename: F
authors.initials: F
authors.lastname: Touri
authors.forename: D
authors.initials: D
authors.lastname: Karamata
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry; D002250:Carbon Radioisotopes; D002473:Cell Wall / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003432:Cross-Linking Reagents; D003960:Diaminopimelic Acid / Q000032:analysis*; D004139:Dinitrofluorobenzene; D006868:Hydrolysis; D010457:Peptidoglycan / Q000737:chemistry*
chemical_list: D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D010457:Peptidoglycan; D003960:Diaminopimelic Acid; D004139:Dinitrofluorobenzene