Misaki Sera

Increasing effect of vanadate on lipoprotein lipase activity in isolated rat fat pads

Vanadate increased lipoprotein lipase (LPL) activity in the isolated fat pads in a time- and dose-dependent manner. The increasing effect of vanadate was inhibited by amiloride, similar to that of insulin, and it also was not additive to that of insulin. Although the increasing effects of vanadate and insulin were preserved in K(+)-free medium, appreciable decreases in both effects were observed by replacement of Na+ with choline ion or omission of Ca2+ in the medium. Vanadate showed the full effect in the presence of cycloheximide at concentrations that inhibited protein synthesis of the fat pads, suggesting that the action of vanadate is not due to the increase in protein synthesis. Tetrakis (acetoxymethyl) ester of quin 2 at 50 microM concentration never inhibited the action of vanadate though it showed a little inhibition at a concentration of 300 microM. No inhibition of the action of vanadate was observed with ruthenium red. These results suggest that vanadate increases the LPL activity via a process less sensitive to the intracellular Ca2+ concentration. Adrenaline, dibutyryl cyclic AMP, and 3-isobutyl-1-methylxanthine all inhibited the action of vanadate, suggesting that the action is inhibited with increase in the intracellular concentration of cyclic AMP. Monensin and carbonyl cyanide m-chlorophenylhydrazone inhibited the action of vanadate. In contrast, the action of insulin was never inhibited by monensin. Tunicamycin and 2-deoxyglucose, at rather high concentrations, inhibited both actions. These findings suggest that vanadate increases the LPL activity through mechanisms of action involving amiloride- and monensin-sensitive pathways dependent on energy.

Stimulatory release of lipoprotein lipase activity from rat fat pads by vanadate.

Vanadate stimulated the release of lipoprotein lipase (LPL) activity from rat fat pads into the medium in a time- and dose-dependent manner. It exerted the synergetic effect with heparin. The stimulatory effects of vanadate and heparin were decreased by incubation in Na+- or Ca2+-free media but were well preserved in K+-free medium. Amiloride inhibited the vanadate-stimulated release of LPL activity in a dose-dependent manner, but did not inhibit the heparin-stimulated release of LPL activity. Colchicine, antimycin A, and carbonyl cyanide m-chlorophenylhydrazone suppressed the stimulatory effect of vanadate, but cycloheximide did not. Preincubation of the fat pads with the tetrakis (acetoxymethyl) ester of quin 2 (quin 2-AM) inhibited the vanadate-stimulatory release of LPL activity without affecting basal activity. The concentration required for half-maximal inhibition of the action of vanadate by quin 2-AM was calculated to be 39 microM, suggesting that the action of vandate was dependent on intracellular Ca2+ concentration. The heparin-stimulated release, on the other hand, was not inhibited even at higher concentrations of quin 2-AM (up to 200 microM). These findings suggest that vanadate stimulates the release of LPL activity through mechanisms of action involving amiloride-sensitive and calcium-dependent pathways with a requirement of metabolic energy.

Electrophoretic and spectroscopic analyses of equine α2-macroglobulin with cleavage of the thiol ester bonds by methylamine

Reaction of equine alpha 2-macroglobulin (alpha 2M) with methylamine caused generation of 3.7 mol of thiol groups per mole of the protein, and the second-order rate constant of the generation was calculated to be 3.5 M-1 s-1. The inhibitory profile of caseinolytic activity of trypsin indicated that one molecule of equine alpha 2M inhibited two molecules of trypsin, similar to human alpha 2M. The methylamine-treated equine alpha 2M, with complete cleavage of the thiol ester bonds, still inhibited the activity of trypsin, though human alpha 2M lost its inhibitory activity by treatment with methylamine. These results indicate that the mode of inhibition of trypsin by equine alpha 2M is substantially unperturbed by cleavage of the thiol ester bonds and that the intact thiol ester bonds per se are not essential for the ability of equine alpha 2M to bind the enzyme. Ultraviolet absorption difference, intrinsic fluorescence, and circular dichroism spectra of the methylamine-treated equine alpha 2M showed that this treatment caused only a small change in conformation of the protein. Reaction of the methylamine-treated protein with trypsin induced appreciable changes in the spectra, indicating a large change in conformation of the protein. These findings were consistent with the results obtained by electrophoresis: The band of methylamine-treated equine alpha 2M showed indistinguishable mobility from that of the unmodified protein, indicating that no appreciable change in conformation occurred, and distinctly different mobility from that of the unmodified or methylamine-treated equine alpha 2M when each had reacted with trypsin.