a-Amylases belong to the alp-barrel protein family in which the active site is created by residues located at the C-terminus of the p strands and in the helix-connecting loops extending from these ends. In the a-amylase family, a small separate domain B protrudes at the C-terminus of the third p strand of the @/a),-barrel framework. The 80% identical barley a-amylase isozymes 1 and 2 (AMYl and AMY2, respectively) differ in substrate affinity and turnover rate, CaC1, stimulation of activity, sensitivity to the endogenous 21-kDa a-amylaselsubtilisin inhibitor, and stability at low pH. To identify regions that confer these isozyme-specific variations, AMY1 -AMY2 hybrid cDNAs were generated by in vivo homologous recombination in yeast. The hybrids AMYl-(l-9O)-AMY2-(90-403) and AMY1-(1-161)-AMY2-(161-403) characterized in this study contain the 90-residue and 161 -residue N-terminal sequences, respectively, of AMY 1 and complementary C-terminal regions of AMY2. AMYl-(1-90)-AMY2-(90-403) comprises the 60-amino-acid domain B of AMY2 and resembles this isozyme in sensitivity to a-amylaselsubtilisin inhibitor and its low affinity for the substrates p-nitrophenyl a-D-maltoheptaoside, amylose and the inhibitor acarbose. Only AMY1-( 1-161)-AMY2-(161-403) and AMYl, which both share domain B, are stable at low pH. However, AMY2 and both hybrid AMY species, but not AMY1, show maximum enzyme activity on insoluble blue starch at approximately 10 mM CaC1,. Domain B thus determines several functional and stability properties that distinguish the barley a-amylase isozymes.a-Amylases hydrolyse internal a-l,4-glucosidic linkages of starch and related dextrins and are widely occurring in microorganisms, higher plants and animals. In cereals, aamylases represent a major starch-degrading activity important in seed germination (MacGregor, 1987). a-Amylases and related amylolytic enzymes are @/a),-barrel proteins (Matsuura et al
Artemisia
has long been used in traditional medicine and as a food source for different functions in eastern Asia.
Artemisia vulgaris
L. (AV) is a species of the genus Artemisia. Essential oils (EOs) were extracted from AV by subcritical butane extraction. EO contents were detected by electronic nose and headspace solid-phase microextraction coupled with gas chromatography (HS-SPME-GC-MS). To investigate the hepatoprotective effects, mice subjected to liver injury were treated intragastrically with EOs or eucalyptol for 3 days. Acetaminophen (APAP) alone caused severe liver injury characterized by significantly increased serum AST and ALT levels, ROS and hepatic malondialdehyde (MDA), as well as liver superoxide dismutase (SOD) and catalase (CAT) depletions. EOs significantly attenuated APAP-induced liver damages. Further study confirmed that eucalyptol is an inhibitor of Keap1, the affinity
K
D
of eucalyptol and Keap1 was 1.42 × 10
−5
, which increased the Nrf2 translocation from the cytoplasm into the mitochondria. The activated Nrf2 increased the mRNA expression of uridine diphosphate glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also inhibiting CYP2E1 activities. Thus, the activated Nrf2 suppressed toxic intermediate formation, promoting APAP hepatic non-toxicity, whereby APAP was metabolized into APAP-gluc and APAP-sulf. Collectively, APAP non-toxic metabolism was accelerated by eucalyptol in protecting the liver against APAP-induced injury, indicating eucalyptol or EOs from AV potentials as a natural source of hepatoprotective agent.
A glycerol-ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight-step purification procedure. The isolation scheme used gave a 483-fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 µmol · min Ϫ1 · mg Ϫ1 . The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size-exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N-terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full-length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol-ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97% identity with that of porcine liver carboxylesterase and more than 50% identity with those of other carboxylesterases from different mammalian species.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.