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Brain and Nerve No to Shinkei Volume 35, Issue 8 （August 1983）

Brain and Nerve 脳と神経 35巻8号（1983年8月発行）

Japanese English

原著

犬脳　phospholipase A₁，A₂およびlysophospholipase活性測定法の微量化と簡略化 MICRO AND SIMPLIFIED ASSAY OF PHOSPHOLIPASE A₁, A₂ AND LYSOPHOSPHOLIPASE ACTIVITIES IN DOG BRAIN 平島豊 ¹ , 甲州啓二 ¹ , 神山和世 ¹ , 遠藤俊郎 ¹ , 高久晃 ¹ , 本田昂 ² , 高崎親久 ³ Yutaka Hirashima ¹ , Keiji Koshu ¹ , Kazuyo Kamiyama ¹ , Shunro Endo ¹ , Akira Takaku ¹ , Takashi Honda ² , Chikahisa Takasaki ³ ¹富山医科薬科大学脳神経外科 ²富山医薬科大学RI施設 ³東北大学理学部化学科 ¹Department of Neurosurgery, Toyama Medical and Pharmaceutical University ²Radioisotope Laboratory, Toyama Medical and Pharmaceutical University ³Department of Chemistry, Faculty of Science, Tohoku University pp.811-817

発行日 1983年8月1日 Published Date 1983/8/1

DOI https://doi.org/10.11477/mf.1406205173

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Abstract 文献概要
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　抄録　脳のphospholipase A₁，　A₂およびlysophospholipase活性測定法に関してはこれまでもその報告がみられるが，いずれも十分精製された酵素に対するものであり，個々の検体間の比較には不適当で，また方法そのものが煩雑であった。今回われわれは，雑種成犬脳を用い上記酵素の抽出法，反応条件，反応生成物の分離等に検討を加え個々の検体間の酵素活性の比較が可能であり，より簡略化されしかも微量化した測定法を開発した。またその再現性の検討，従来法との比較では，再現性も良くしかも従来法より若干高い値を示すなど有利な点が証明された。

The purpose of our study was to examine the ischemia induced enzymatic changes of decaylation-reacylation cycle of membrane phospholipids in dog brain.

In this study, we developed new modified me-thod for assay of phospholipase A₁, A₂ and lyso-phospholipase which is simpler and needs only a smaller amount of materials. For the first report, we introduced this new method and demonstrated some properties of phospholipase A₁, A₂ and lyso-phospholipase in dog brain.

Crude enzyme solution for assays of phospholi-pase A₁, A₂ and lysophospholipase was gained from extraction of frozen brain with aceton, bu-tanol and saline. The level of phosphorus in the enzyme extract was determined and only those extracts which had a level of phosphorus within a certain range were used. The substrates for assays were L-α-〔β-palmitoyl-1-¹⁴C〕 phosphati-dylcholine, dipalmitoyl for phospholipase A₁ and A₂ and L-lysophosphatidylcholine-1-〔1-¹⁴C〕 pal-mitoyl for lysophospolipase respectively. Each ra-dioactive substrates was diluted with cold carrier lipid to give the proper specific activity. Reaction system including substrate, buffer (pH 7. 0) and enzyme extract was incubated for 10 hours at 38 ℃. But for the assay of phospholipase A₁ and A₂, enzyme solution was pre-incubated at 70℃ for 5 minutes. In our new method, reaction mixture was directly separated by TLC without extracting li-pids. Enzyme activities were calculated from radio thin-layer chromatograms.

Furthermore, we made a comparison between our method and the former one. The value of each enzyme activity was slightly higher in our method than in the former one. However, it was revealed that the results were reproducible in both me-thods.