The active streptococcal hyaluronan synthases (HASs) contain a single HAS monomer and multiple cardiolipin molecules.
The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radiation inactivation analysis of isolated membranes. The native enzymes in membranes from Group A Streptococcus pyogenes HAS and Group C Streptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia coli membranes. Based on their amino acid sequences, the masses of these four proteins as monomers are approximately 48 kDa. In all cases, loss of enzyme activity was a simple single exponential function with increasing radiation dose. The functional sizes calculated from these data were identical for the four HASs at approximately 64 kDa. In contrast, the sizes of the proteins estimated by the loss of antibody reactivity on Western blots were essentially identical at 41 kDa for the four HAS species, consistently lower than the functional size by approximately 23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H6 and S. equisimilis HAS-H6 gave masses that differed by <0.07% from the predicted monomer sizes, which confirms that neither protein is posttranslationally modified or covalently attached to another protein. Ongoing studies indicate that the purified HAS enzymes require cardiolipin (CL) for maximal activity and stability. When irradiated membranes were detergent solubilized and the extracts were incubated with exogenous CL, the residual level of HAS activity increased. Consequently, the calculated functional size decreased by approximately 23 kDa to the expected size of the HAS monomer. The approximately 23-kDa larger size of the functional HAS enzyme, compared with the HAS monomer, is due, therefore, to CL molecules. We propose that the active streptococcal HA synthases are monomers in complex with approximately 16 CL molecules.
Tlapak-Simmons
V L
VL
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Kempner
E S
ES
Baggenstoss
B A
BA
Weigel
P H
PH
D002308:Cardiolipins / Q000737:chemistry*; D004307:Dose-Response Relationship, Radiation; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000528:radiation effects; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry; D008970:Molecular Weight; D011839:Radiation, Ionizing; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013291:Streptococcus / Q000201:enzymology*; D014166:Transferases; D029867:Xenopus Proteins
D002308:Cardiolipins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases