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An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation.
Clin Chim Acta. 1978 Jun 15; 86(3):301-5.CC

Abstract

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.

Authors

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Pub Type(s)

Journal Article

Language

eng

PubMed ID

668120

Citation

Schutgens, R B., et al. "An Improved Quantitative Assay of Galactose-1-phosphate Uridyltransferase Activity in Erythrocytes Based On the Determination of Glucose 1-phosphate Generation." Clinica Chimica Acta; International Journal of Clinical Chemistry, vol. 86, no. 3, 1978, pp. 301-5.
Schutgens RB, Berntssen WJ, Pool L. An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation. Clin Chim Acta. 1978;86(3):301-5.
Schutgens, R. B., Berntssen, W. J., & Pool, L. (1978). An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation. Clinica Chimica Acta; International Journal of Clinical Chemistry, 86(3), 301-5.
Schutgens RB, Berntssen WJ, Pool L. An Improved Quantitative Assay of Galactose-1-phosphate Uridyltransferase Activity in Erythrocytes Based On the Determination of Glucose 1-phosphate Generation. Clin Chim Acta. 1978 Jun 15;86(3):301-5. PubMed PMID: 668120.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation. AU - Schutgens,R B, AU - Berntssen,W J, AU - Pool,L, PY - 1978/6/15/pubmed PY - 1978/6/15/medline PY - 1978/6/15/entrez SP - 301 EP - 5 JF - Clinica chimica acta; international journal of clinical chemistry JO - Clin Chim Acta VL - 86 IS - 3 N2 - An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C. SN - 0009-8981 UR - https://www.unboundmedicine.com/medline/citation/668120/An_improved_quantitative_assay_of_galactose_1_phosphate_uridyltransferase_activity_in_erythrocytes_based_on_the_determination_of_glucose_1_phosphate_generation_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0009-8981(78)90385-6 DB - PRIME DP - Unbound Medicine ER -