Fig 1.
AGLA activity in lymphoblastoid cells.
Cell lines GM7027 (male) and GM7053 (female) were grown in FA free media for three days prior to incubating with the indicated FA concentrations in RPMI 1640 medium. After three days, cells were collected by centrifugation, rinsed with normal saline, and AGLA activity determined in presence of NAG. Values are means of three independent experiments and bars show SEM. AGLA values at each FA concentration was significantly different (P < 0.0001) from that at zero concentration.
Fig 2.
FA supplementation stimulates AGLA expression in lymphoblastoid cells at transcript and protein levels.
Cells were grown in FA free medium for three days prior to incubating with the indicated FA concentrations in RPMI 1640 medium. After three days, cells were collected by centrifugation, rinsed with normal saline, and RNA or proteins were prepared. (A) Transcript levels were measured by RT-PCR using primer pair HP200154 for GLA and HP200179 for HPRT1. Values are mean of three independent experiments and bars show the SEM. Differences among concentrations were significant (F (5,12) = 3.85, p < 0.026). (B) Western blot analysis of lymphoblastoid cells exposed to FA concentrations, (25 µg protein each) were probed with an anti-AGLA rabbit monoclonal antibody. Picture of a representative blot. (C) Densitometric measurement of blots using Image J, and expression of ratio with the housekeeping gene GAPDH. Differences among concentrations were extremely significant (F (5,12) = 19.81, p < 0.0001).
Fig 3.
Stimulation of recombinant AGLA by FA.
Recombinant AGLA (Accession # P06280) activity was determined in the presence of indicated FA concentration using 4-MU-α-Gal as the substrate. Release of 4-MU was determined fluorometrically. Values represent means with bars showing SEM for three independent measurements. AGLA activity was significantly different (P < 0.05) at all concentrations when measured against zero FA.
Fig 4.
Kinetic properties of recombinant AGLA in the presence of FA.
AGLA activity was determined at various 4-MU-α-Gal concentrations at indicated FA concentration. Km (A) and Vmax (B) were determined from double reciprocal Lineweaver-Burk plots. Values are means and bars show SEM of three independent measurements.
Table 1.
FA preincubation with AGLA is beneficial.
Fig 5.
FA reverses DGJ induced inhibition of recombinant AGLA activity.
Recombinant AGLA was incubated with or without the specific inhibitor DGJ, either in the presence or absence of FA at varying concentrations of the substrate, 4MU-Gal (A). Fluorescence was measured after terminating the reaction with the stop solution. (B) Lineweaver-Burk plot of the data to determine type of enzyme mechanism.
Fig 6.
FA stimulates DGJ mediated rescue of AGLA activity in lymphoblastoid cells from a FD individual.
AGLA activity in the cell line GM04391 established from a FD individual was measured without or with DGJ (7 µM), at indicated concentrations of FA in the media. Values represent mean ± SEM of three independent experiments.