geoscience.net logo
+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate


, : A sensitive high-performance liquid chromatographic-fluorometric assay for dihydrofolate reductase in adult rat brain, using 7,8-dihydrobiopterin as substrate. Analytical Biochemistry 140(2): 548-552

A liquid chromatographic-fluorometric assay has been developed to study the role of dihydrofolate reductase in adult rat brain since low levels of the enzyme preclude measurement by current spectrophotometric procedures. This method involves in vitro incubation of desalted, cell-free brain extracts with 7,8-dihydrobiopterin, NADPH, and an NADPH-regenerating system. The tetrahydrobiopterin formed is quantitatively converted to pterin using alkaline iodine oxidation, and the pterin formed is separated by liquid chromatography and detected fluorometrically. The method is linear from 100 fmol to greater than or equal to 1 nmol of product, and the sensitivity is at least 100 times greater than that of existing spectrophotometric assays. Enzyme activity of desalted brain extracts is linear with both time (to 100 min) and protein (from 50 to 620 micrograms). The enzyme shows an absolute requirement for NADPH, does not use NADH, and is completely inhibited by 10 nM methotrexate. The Km of the enzyme for NADPH was found to be 7.5 microM, while the Km for 7,8-dihydrobiopterin was 88 microM. Since brain dihydrobiopterin reductase has the same properties as dihydrofolate reductase, this fluorometric procedure can serve as a sensitive assay for dihydrofolate reductase.

US$29.90

PMID: 6486439


Other references

Henderson, J.T.; Chopko, A.L.; van Wassenaar, P.D., 1992: Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. The plasmid-encoded bacteriocin pediocin PA-1, produced by the gram-positive bacterium Pediococcus acidilactici strain PAC-1.0, was purified to homogeneity. The purified product exhibited antibacterial activity against several gram-positive bacter...

Kitaev, A.; Preskill, J., 2006: Topological entanglement entropy. We formulate a universal characterization of the many-particle quantum entanglement in the ground state of a topologically ordered two-dimensional medium with a mass gap. We consider a disk in the plane, with a smooth boundary of length L, large c...

Blaine, G.; Oliver, L.C., 1952: A simplified one-stage method of cranioplasty with acrylic resin. British Journal of Surgery 39(156): 371-372

Podolsky S., 1986: The diabetic lipodystrophies. Jelinek, J E (Ed ) The Skin in Diabetes Xiii+237p Lea And Febiger: Philadelphia, Pa , Usa Illus 203-216

Kennedy, D.W., 2012: Radiological localization of Schneiderian papilloma. International Forum of Allergy & Rhinology 1(6): 492-492

Loew, R.; Vigna, E.; Lindemann, D.; Naldini, L.; Bujard, H., 2006: Retroviral vectors containing Tet-controlled bidirectional transcription units for simultaneous regulation of two gene activities. In this study retroviral self-inactivating (SIN)-vectors were constructed, that allow simultaneous regulation of two genes by integration of bidirectional Tet controlled transcription units. Marker genes (luciferase and eGFP) were expressed under...

Liu, H.L.ng; Gao, J.H.ng, 1999: Perfusion-based event-related functional MRI. Perfusion-based event-related functional MRI was performed by measuring flow-sensitive alternating inversion recovery (FAIR) signal changes during repeated single-trial, short visual stimulation (250 msec). In the visual cortex activation area, th...

Charoenkul, V.; Giron, F.; Peirce, E.C., 1973: Respiratory support with a paracorporeal membrane oxygenator. Journal of Surgical Research 14(5): 393-399

Bánóczy, Ján.; Rugg-Gunn, A.J., 2007: Caries prevention through the fluoridation of milk. A review. The aim of this review is to give an overview of 50 years experience of milk fluoridation and draw conclusions about the applicability of the method. Fluoridated milk was first investigated in the early 1950s, almost simultaneously in Switzerland,...

Hilger, Hh, 1986: Flower and fruit development in Wigandia caracasana (Hydrophyllaceae). American journal of botany 74(2): 250-259