Size of the catalytically active unit of rat hepatic carboxylester lipase in the presence and absence of bile salt.

Carboxylester lipase (CEL) catalyzes the hydrolysis of cholesteryl esters, retinyl esters, and triacylglycerols. CEL monomer has a MW of approximately 70000. Hydrolysis of these esters is stimulated by millimolar trihydroxy bile salts such as cholate, that also induce aggregation. Liver cytosols from 12 rats were frozen and irradiated at -135 degrees C with high energy electrons. In several experiments, paired samples of cytosol were adjusted to 20 mM cholate before irradiation. All samples were assayed for CEL using cholesteryl oleate as substrate. In untreated cytosols, CEL activity surviving radiation exposure could be fit to a single exponential function, the slope of which yielded a target size of 91 +/- 18 kDa. In a subset of these cytosols irradiated in the presence of cholate the calculated target size was 100 +/- 19 kDa, a value indistinguishable from that obtained for untreated cytosols. Some samples were also assayed using retinyl palmitate and triolein as substrates. With retinyl palmitate the mean target sizes were 96 and 108 kDa in the absence and presence of cholate, respectively, approximately the same as those observed when using cholesteryl oleate. When triolein was used as substrate the target sizes in the absence of cholate were smaller than for the other two esters (67 +/- 18 kDa) and closer to the known monomer molecular weight, but again cholate had no significant effect on this size. The structure responsible for CEL activity contains no more than one 70000 MW monomer and the results show that cholate-induced oligomerization is not required for catalytic activity.

Characterization and inhibition of 15-lipoxygenase in human monocytes: comparison with soybean 15-lipoxygenase.

These studies characterized a method to measure 15-lipoxygenase (15-LO) activity in human monocytes (HMC) exposed to interleukin-4 (IL-4) and compare the activity with that of soybean 15-LO. 15-LO activity was quantitated by measuring 15-[14C]hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) production from the substrate [14C]arachidonic acid (AA) after high-performance liquid chromatographic or thin-layer chromatographic separation. 15-HETE production by HMC was significantly elevated after continuous exposure to a single dose (10 ng/ml) of IL-4 for 4 days, was maximal at 5 days, and remained elevated at 6 days. At 6 days 15-LO activity in IL-4-treated HMC was increased significantly (2.81 +/- 0.70 fmol 15-HETE/cell) compared with background levels of 15-HETE in untreated HMC (0.098 +/- 0.31 fmol 15-HETE/cell). 15-HETE production was linear in the range of 5 x 10(4) to 7 x 10(5) HMC/assay, from 2 to 160 microM AA, and during 5-10 min of incubation with AA. Ethyl 2-cyano-3-(3',4'-dihydroxyphenyl)propenoate (a caffeic acid analogue), N-methyl-4-benzyloxy-phenyl acetohydroxamate (RG-6866), esculetin, and four novel lipoxygenase inhibitors, a phenylcyanomethylene analogue (RP-27493) and three benzoxadiazine analogues (RP-64835, RP-65047, and RP-65208), inhibited HMC 15-LO, with concentrations eliciting 50% of maximal inhibition (microM) of 0.21, 0.8, 6.3, 10, 22, 20, 6.3, 3, and 20 and 5.8, 5, 12, 1, 30, 0.8, 0.15, 0.05, and 2.8 for inhibition of soybean 15-LO, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile salt-dependent and bile salt-independent cholesteryl ester hydrolase activities in rat liver cytosol.

These studies report on the relationship between the bile salt-dependent and -independent cholesteryl ester hydrolase (CEH) activities found in rat liver cytosol. The two activities show very similar Michaelis-Menten substrate kinetics and pH dependence. After gel filtration of cytosol, the bile salt-independent activity elutes much earlier than the bile salt-dependent activity, suggesting that the two activities are associated with entities of different molecular size. However, when gel filtration is carried out in the presence of bile salt, the bile salt-dependent activity elutes as a large aggregate, similar to the bile salt-independent activity's behavior in the absence of bile salt. Both activities coelute after cytosol is passed through an ion exchange column. After each chromatographic procedure the recovery of the bile salt-dependent activity was substantially higher than the recovery of the bile salt-independent activity. When cytosol is incubated with anti-rat pancreatic CEH in the absence of cholate, the bile salt-dependent activity is inhibited more than 90% whereas bile salt-independent activity remains unaffected even at high antibody concentrations. When cytosol is incubated with anti-rat pancreatic CEH in the presence of cholate both CEH activities remain unaffected. The prevention of immunoinhibition by cholate seems to be specific for this detergent since CHAPS, a cholate analog, does not prevent immunoinhibition of the bile salt-dependent activity by anti-CEH. The experimental results are consistent with a model for CEH activity in liver cytosol in which there is only one enzyme that can exist in a monomeric, inactive form (that can be activated by addition of cholate to the assay and represents the bile salt-dependent activity) and in an active complex comprising several enzyme monomers as well as cholate micelles (that accounts for the bile salt-independent activity).

Analysis of microsomal cholesteryl ester hydrolases by radiation inactivation.

Radiation inactivation by high energy electrons, a method for determining the size of a protein without prior purification, was used to study the acid and neutral cholesteryl ester hydrolase (CEH) activities of rat liver microsomes. The same preparations were also assayed for the microsomal, "nonspecific" carboxylesterases using o-nitrophenyl acetate as substrate. Non-specific esterase activity surviving radiation could be fit to a single exponential function, the slope of which yielded a target size of 47 +/- 5 kDa (mean +/- S.D., n = 7). Surviving CEH activity assayed at pH 5 could also be fit to a single exponential that yielded a target size of 71 +/- 14 kDa (n = 5). In contrast, the surviving CEH activity assayed at pH 7 was more complex. The data from six experiments were described as the sum of two exponentials, indicating that most of the activity is due to an entity that is three to four times larger and a minor amount to one that is half the size of the pH 5 enzyme. The results are consistent with the suggestion that the acid and neutral microsomal CEH activities are due to distinct enzymes, which are not the "nonspecific" carboxylesterases. Their sizes also differ from those previously determined for lysosomal acid lipase and other lipases in the liver.