PMID- 10348910
OWN - NLM
STAT- MEDLINE
DCOM- 19991108
LR  - 20190513
IS  - 0021-924X (Print)
IS  - 0021-924X (Linking)
VI  - 125
IP  - 6
DP  - 1999 Jun
TI  - Purification and characterization of diacylglycerol lipase from human platelets.
PG  - 1077-85
AB  - Diacylglycerol lipase (DGL) was solubilized from human platelet microsomes with
      heptyl-beta-D-thioglucoside, and purified to homogeneity on SDS-PAGE using a
      combination of chromatographic and electrophoretic methods. The molecular mass of
      the purified DGL was estimated to be 33 kDa. Its apparent pI was pH 6.0, as
      determined by Immobiline isoelectro-focusing. The enzymatic activity of the
      partially purified DGL was investigated in the presence of a variety of
      inhibitors and reagents, as well as its pH and calcium dependence. Thiol reagents
      such as p-chloromercurubenzoic acid (pCMB), N-ethylmaleimide (NEM), and HgCl2
      inhibited the activity, while dithiothreitol (DTT) and reduced glutathione (GSH) 
      enhanced it. In addition, the enzymatic activity was inhibited by two serine
      blockers, phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate
      (DFP), and by a histidine modifying reagent, p-bromophenacyl bromide (pBPB).
      These results suggest that cysteine, serine and histidine residues are required
      for the enzymatic activity of DGL. DGL was optimally active in the pH range of
      7-8 and its activity did not change significantly in the presence of various
      calcium concentrations, even in the presence of 2 mM EGTA. This indicates that
      DGL can hydrolyze substrates with a basal cytosolic free Ca2+ level in the
      physiological pH range. A DGL inhibitor, RHC-80267, inhibited DGL activity in a
      dose-dependent manner with an IC50 (the concentration required for 50%
      inhibition) of about 5 microM. Unexpectedly, several phospholipase A2 (PLA2)
      inhibitors were potent inhibitors of DGL activity (IC50<5 microM), suggesting
      that the catalytic mechanisms of DGL and PLA2 may be similar. Finally, we show
      that DGL activity was inhibited by 2-monoacylglycerols (2-MGs), the reaction
      products of this enzyme. Among the three 2-MGs tested (2-arachidonoyl glycerol,
      2-stearoyl glycerol, and 2-oleoyl glycerol), 2-arachidonoyl glycerol was the most
      potent inhibitor.
FAU - Moriyama, T
AU  - Moriyama T
AD  - Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011,
      Japan. moriyama@soya.food.kyoto-u.ac.jp
FAU - Urade, R
AU  - Urade R
FAU - Kito, M
AU  - Kito M
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - J Biochem
JT  - Journal of biochemistry
JID - 0376600
RN  - 0 (Detergents)
RN  - 0 (Enzyme Inhibitors)
RN  - EC 3.1.1.34 (Lipoprotein Lipase)
SB  - IM
MH  - Blood Platelets/*enzymology
MH  - Catalytic Domain
MH  - Detergents
MH  - Enzyme Inhibitors/pharmacology
MH  - Humans
MH  - In Vitro Techniques
MH  - Isoelectric Point
MH  - Lipoprotein Lipase/blood/chemistry/*isolation & purification
MH  - Microsomes/enzymology
MH  - Molecular Weight
MH  - Solubility
EDAT- 1999/06/01 00:00
MHDA- 1999/06/01 00:01
CRDT- 1999/06/01 00:00
PHST- 1999/06/01 00:00 [pubmed]
PHST- 1999/06/01 00:01 [medline]
PHST- 1999/06/01 00:00 [entrez]
AID - 10.1093/oxfordjournals.jbchem.a022389 [doi]
PST - ppublish
SO  - J Biochem. 1999 Jun;125(6):1077-85. doi: 10.1093/oxfordjournals.jbchem.a022389.