{"created":"2025-04-23T08:49:50.363838+00:00","id":2000177,"links":{},"metadata":{"_buckets":{"deposit":"3aa33ae8-ebfd-49b7-a74e-656466657b15"},"_deposit":{"created_by":15,"id":"2000177","owner":"15","owners":[15],"pid":{"revision_id":0,"type":"depid","value":"2000177"},"status":"published"},"_oai":{"id":"oai:kjunshin.repo.nii.ac.jp:02000177","sets":["1712807540574:1739761729484"]},"author_link":["1298"],"control_number":"2000177","item_2_biblio_info_14":{"attribute_name":"bibliographic_information","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2025-03","bibliographicIssueDateType":"Issued"},"bibliographicIssueNumber":"29","bibliographicNumberOfPages":"8","bibliographicPageEnd":"31","bibliographicPageStart":"24","bibliographicVolumeNumber":"-","bibliographic_titles":[{"bibliographic_title":"鹿児島純心大学看護栄養学部紀要","bibliographic_titleLang":"ja"}]}]},"item_2_creator_6":{"attribute_name":"著者名(日)","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"南, 勇真","creatorNameLang":"ja-Kana"}],"familyNames":[{"familyName":"南","familyNameLang":"ja-Kana"}],"givenNames":[{"givenName":"勇真","givenNameLang":"ja-Kana"}],"nameIdentifiers":[{"nameIdentifier":"1298","nameIdentifierScheme":"WEKO"},{"nameIdentifier":"1530300152483168640","nameIdentifierScheme":"CiNii ID","nameIdentifierURI":"http://ci.nii.ac.jp/nrid/1530300152483168640"}]}]},"item_2_description_12":{"attribute_name":"item_2_description_12","attribute_value_mlt":[{"subitem_description":"【Introduction】Enzymes play a pivotal role in living organisms by catalyzing a variety of essential reactions. It is particularly important for students involved in nutrition or biochemistry to learn about enzymes and their properties from a chemical perspective. On the other hand, it is difficult to perform rigorous measurements of enzyme activity at the student experimental level. This bulletin article presents simple experiments and analyses for understanding the Michaelis-Menten equation.\n【Methods】The hydrolysis of phosphate by alkaline phosphatase (ALP) was used as a model reaction. Commercially available ALP from E.coli. was used for the dephosphorylation of p-nitrophenyl phosphate (p-NPP) to p-nitrophenol (p-NP). The reaction progress was monitored by measuring the absorbance at 420 nm derived from p-NP under alkaline conditions. Inorganic phosphate was used as an ALP inhibitor. The concentration of the product (p-NP) was estimated from a calibration curve, and approximate reaction rates were also calculated. Based on Michaelis-Menten equation, Vmax and Km were also obtained in each reaction conditions. All data were analyzed using Excel.\n【Results and Discussion】A calibration curve for p-NP was constructed, demonstrating strong linearity based on Lambert-Beer’s law, enabling precise quantification of reaction products. The reaction rate was highest within the first minute and decreased over time, with the maximum concentration of p-NP (100 µM) reached within 15 minutes. The Michaelis-Menten kinetics of ALP were analyzed, yielding Vmax = 0.0254 mM/min, and Km = 0.0290 mM from a Lineweaver-Burk plot. The inhibitory effect of inorganic phosphate (Pi) was assessed by introducing two concentrations of Pi (0.1 and 0.2 mM). Analysis of the Lineweaver-Burk plots showed increased slopes with constant y-intercepts, indicating competitive inhibition. This was consistent with a mode of inhibition in which the inhibitor binds to the enzyme’s active site, increasing Km without altering Vmax. These results provide insights into the kinetic properties of ALP and the competitive inhibition exerted by its reaction byproduct, inorganic phosphate.\n【Summary】In this report, protocols for ALP activities measurements were introduced for student experiments. The measurements are based on spectroscopic analysis and Michaelis-Menten equation. These experiments need no special equipment or software and are easy to perform. The Lineweaver-Burk plot of experimental results shows good accordance with representative competitive inhibition. Experiments on other forms of inhibition could be conducted by changing the inhibitor.","subitem_description_language":"en","subitem_description_type":"Abstract"}]},"item_2_relation_13":{"attribute_name":"item_2_relation_13","attribute_value_mlt":[{"subitem_relation_type":"isVersionOf","subitem_relation_type_id":{"subitem_relation_type_id_text":"-","subitem_relation_type_select":"Local"}}]},"item_2_source_id_13":{"attribute_name":"item_2_source_id_13","attribute_value_mlt":[{"subitem_source_identifier":"2759-050X","subitem_source_identifier_type":"ISSN"}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2025-04-23"}],"filename":"Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation for Advanced Student Experiments.pdf","filesize":[{"value":"1.8 MB"}],"format":"application/pdf","licensetype":"license_5","mimetype":"application/pdf","url":{"url":"https://kjunshin.repo.nii.ac.jp/record/2000177/files/Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation for Advanced Student Experiments.pdf"},"version_id":"7cb19912-05db-4ad6-ac5e-35caa32f4d3c"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"item_resource_type","attribute_value_mlt":[{"resourcetype":"departmental bulletin paper","resourceuri":"http://purl.org/coar/resource_type/c_6501"}]},"item_title":"Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation　for Advanced Student Experiments","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation　for Advanced Student Experiments","subitem_title_language":"en"}]},"item_type_id":"2","owner":"15","path":["1739761729484"],"pubdate":{"attribute_name":"公開日","attribute_value":"2025-04-23"},"publish_date":"2025-04-23","publish_status":"0","recid":"2000177","relation_version_is_last":true,"title":["Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation　for Advanced Student Experiments"],"weko_creator_id":"15","weko_shared_id":-1},"updated":"2025-04-23T09:18:10.560661+00:00"}