Tissue Distribution Of Activity Research Articles

Purification, Identification, and Cloning of Lysoplasmalogenase, the Enzyme That Catalyzes Hydrolysis of the Vinyl Ether Bond of Lysoplasmalogen

Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent Km values of ∼50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent Vm values of 24.5 and 17.5 μmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (Ki ∼20 μm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells.

Aldrin epoxidation and dihydroisodrin hydroxylation as probes of in vivo and in vitro oxidative metabolic capability of some caterpillars

Comparative biochemical studies are productive means to study factors that limit both beneficial and harmful effects of chemicals. Reactions such as aldrin epoxidation and dihydroisodrin hydroxylation are valuable assays of oxidative metabolism in scientific studies of chemical biology in insects, subhuman primates and other living things. The tissue distribution of activity in caterpillars may have functional significance. Localization of relatively high concentrations of these cytochrome P450 monooxygenases in gut tissue of lepidoptera may represent an important means to minimize absorption of lipophilic foreign chemicals in food. Some polychlorocycloalkanes permit in vivo and in vitro studies owing to their stability, acceptable toxicity and relatively simple pattern of metabolism. In vivo studies to assess the significance of in vitro findings are feasible with substrates such as aldrin, dihydroisodrin (DHI) and oxidative methylenedioxyphenyl inhibitors such as piperonyl butoxide (PBO) or carbon monoxide. Biphasic dose-dependent decreased and increased DHI-OH formation resulted from PBO pretreatment by gut, fat body, head and Malpighian tubule homogenates of cutworms and gut and fat body (the only tissues tested) of cabbage looper Trichplusia ni (Hübner) and black cutworm Agrotis ipsilon (Hüfnagel). The biphasic in vivo responses of caterpillars to PBO are a reminder of the complexity of biochemical and physiological responses of organisms coexposed to chemicals that are classified, often glibly, as toxic substances and metabolic inhibitors and inducers. Knowledge of dose and time relationships demands very careful evaluation in living things in the environment.

Resorption rate, route, route of elimination, and ultrastructure of the implant site of polylactic acid in the abdominal wall of the rat.

AbstractThis experiment describes resorption rate, light and electron microscopic appearance of the implant site, and tissue distribution and route of elimination of radioactivity from carbon‐labelled polylactic acid implants. Fifty‐six rats received abdominal implants and were serially sacrificed at 28, 56, 84, 112, 140, 154, and 168 days. Urine and fecal output was measured for radioactivity, along with implant site and several tissues at the time of sacrifice. Tissue distribution of activity was negligible. Resorption rate of the implant was linear with 63.2% remaining after 168 days. Elimination of radioactivity was via respiration, approximately 29.4% by this route. Histologically, the implant was invaded rapidly along septal defects or slowly on broad fronts. Invading cells were characterized either by large double‐walled vacuoles or by large accumulations of mitochondria and microvesicles adjacent to the implant surface. The long duration of this type of implant may be an important consideration in surgical applications.