PREPARATION OF GLUCOSE-6-PHOSPHATE UNIFORMLY LABELED WITH $sup 14$C (in Italian)
Uniformly labeled glucose was converted by yeast hexokinase to uniformly labeled glucose-6-phosphate and separated from fructose-6-phosphate and other products by ion-exchange chromatography. Thirty-one ml of a solution at pH 8.2 containing 25.2 mg of uniformly labeled glucose (0.5 mC), 125 mg of ATP Na salt, approximates 2 mg of hexokinase, and MgCl/sub 2/ (0.01M) was incubated 2 hr at 37 deg C. It was brought to 250 ml with H/sub 2/O and poured on a column (1.3-cm dia, 15-cm height) of 20 ml Dowex 1-X8, Cl/sup -/, 200 to 400 mesh, at a rate of 0.5 ml/min. Then the column was washed with 20 ml H/sub 2/O. Elution of the sample was carried out at a rate of 1 to 2 ml/min using a series of solutions (a: 0.001M NH/sub 4/OH; b: 0.025M NH/sub 4/Cl, 0.01M K/sub 2/B/sub 4/O/sub 7/; c: 0.035M NH/sub 4/Cl, 0.001M K/sub 2/B/sub 4/O/sub 7/ 0.0025M NH/sub 4/OH; d: 0.04M NH4Cl, 0.0000lM K/sub 2/B/sub 4/O/sub 7/, 0.0025M NH/sub 4/OH; e: 1N HCl) described by Longinotti et al. Glucose-6-phosphate was eluted by solvent c; fructose-6-phosphate by solvent d; the radiometric chromatogram is illustrated. The products were pUrified by combining the fractions of each separately and then diluting 1: 1 with H/sub 2/O. The solution was set on a column (1.7-cm dia, 25- cm height) of 55 ml Dowex 1-X8, HCOO/sup -/, 200 to 400 mesh, at a rate of 0.5 to 1 ml/min. The column was washed with approximates 100-ml 0.0001M HCOOH and the compound eluted with 2 1 0.7N HCOOH at 1 ml/min. The fractions containing the radioactive peak were combined, and evaporated on a rotary evaporator (30 to 35 deg C) to a small volume. Then 50 ml H/sub 2/O was added and the process repeated three times, the sample finally being dried over P/sub 2/O/sub 5/ and KOH and extracted three times with 20 ml ethyl ether. Evaporation of the ether gave pure phosphate, the G-6-P containing 2405/4200 of the original activity and the F-6-P containing 425/4200 (expressed counts/min x 10/sup -3/). (BBB)
- Research Organization:
- Centro Internazionale di Biochimica Generale e Microbiologia, Rome
- NSA Number:
- NSA-18-017857
- OSTI ID:
- 4049205
- Journal Information:
- Gazzetta Chimica Italiana (Italy), Vol. Vol: 93; Other Information: Orig. Receipt Date: 31-DEC-64
- Country of Publication:
- Country unknown/Code not available
- Language:
- Italian
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Related Subjects
ACIDITY
ADENINE
ADENOSINE
AMMONIA
AMMONIUM COMPOUNDS
ANIONS
ATP
BORATES
CARBON 14
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
CHROMATOGRAPHY
CONFIGURATION
DIAGRAMS
DOWEX
EFFICIENCY
ENZYMES
ESTERS
ETHERS
ETHYL RADICALS
EVAPORATION
EXTRACTION COLUMNS
FORMIC ACID
FRUCTOSE
GLUCOSE
GRAIN SIZE
HEXOKINASE
HEXOSE
HYDROCHLORIC ACID
IMPURITIES
ION EXCHANGE
ION EXCHANGE MATERIALS
KINASES
LABELLED COMPOUNDS
LABORATORY EQUIPMENT
LIQUID FLOW
MAGNESIUM CHLORIDES
MOLECULES
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
OPERATION
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PHOSPHATES
PHOSPHORUS OXIDES
POTASSIUM COM