Role of 2,6-dideoxy-2,6-diaminoglucose in activation of a eukaryotic phospholipase C by aminoglycoside antibiotics.
Recent emergence of microbial resistance to aminoglycoside antibiotics, and the documented cytotoxicity associated with their use, calls for sustained efforts at understanding the effects of the compounds on eukaryotic cells. Using a glycosyl phosphatidylinositol (GPI)-phospholipase C (GPI-PLC) from the protozoan parasite Trypanosoma brucei, we demonstrate that a eukaryotic PLC can be activated 6-fold by aminoglycosides. Neomycin B protected GPI-PLC from a reduction in activity at pH 6.5, and increased the turnover number (kcat) of the enzyme. In structure-activity studies with the neomycin group, 2-deoxy-streptamine was mildly stimulatory; the concentration required to activate GPI-PLC 2-fold (SC200) was 310 microM. Neamine was 150-fold more active (SC200 = 2 microM) than 2-deoxy-streptamine, indicating that a 2,6-dideoxy-2, 6-diaminoglucose substituent at the 4-position of 2-deoxystreptamine plays an important role in activation of GPI-PLC. Ribostamycin and neomycin B also had SC200's of 2 microM, implying that the ribose group in ribostamycin is not involved in activation of GPI-PLC. These conclusions were affirmed in studies with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. A 2, 6-dideoxy-2,6-diaminoglucose substitution at the 4-OH of 2-deoxystreptamine activates the enzyme 17-fold, while a second 2, 6-dideoxy-2,6-diaminoglucose moiety on the ribose ring of ribostamycin provides an additional 3.5-fold stimulation. Possible implications of these observations for the effects of aminoglycosides on eukaryote cells are discussed.
Morris
J C
JC
Department of Cellular Biology, University of Georgia, Athens, Georgia 30602, USA.
Mensa-Wilmot
K
K
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001413:Bacillus thuringiensis / Q000201:enzymology; D004789:Enzyme Activation; D005601:Framycetin / Q000494:pharmacology; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000201:enzymology; D010738:Type C Phospholipases / Q000737:chemistry / Q000378:metabolism*
D000900:Anti-Bacterial Agents; C038761:neosamine C; D005601:Framycetin; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054800:Glycosylphosphatidylinositol Diacylglycerol-Lyase; D005944:Glucosamine