Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

Betts, S A; Mayer, R. John

doi:10.1042/bj1510263

Cited by 33 publications

(20 citation statements)

References 15 publications

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“…per animal) were removed. The particle-free supernatant was prepared by a modification of the method of Betts & Mayer (1975). The tissue was homogenized in 2vol.…”

Section: Assay Of Acetyl-coa Car4oxylasementioning

confidence: 99%

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Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland

Manning¹,

Dils²,

Mayer³

1976

Biochemical Journal

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1. Acetyl-Coa carboxylase from lactating-rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. Use of phosphate buffer throughout the purification gave low recovery of enzyme. Consequently, Tris buffers were used in the extraction and in selected stages of the purification procedure. 3. The purified enzyme had a specific activity of 5.15 +/- 0.3 μmol of bicarbonate incorporated/min per mg of protein (mean +/- S.E.M. of five preparations). This represents a purification of 257 +/- 16-fold and a yield of 4.3 +/- 0.13%. 4. The kinetic parameters of the purified enzyme were similar to those reported for the enzyme from other tissue sources. 5. The enzyme was assayed by a spectrophotometric assay and by a [14C]bicarbonate-fixation assay. Short incubation were used in the radio-chemical assay to avoid substantial loss of [14C]bicarbonate.

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“…per animal) were removed. The particle-free supernatant was prepared by a modification of the method of Betts & Mayer (1975). The tissue was homogenized in 2vol.…”

Section: Assay Of Acetyl-coa Car4oxylasementioning

confidence: 99%

“…Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate Polyacrylamide disc-gel electrophoresis of protein samples, including subsequent staining for protein and destaining, was performed by the method of Betts & Mayer (1975) with 5 % (w/v) polyacrylamide gels.…”

Section: Protein Determinationmentioning

confidence: 99%

Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland

Manning¹,

Dils²,

Mayer³

1976

Biochemical Journal

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“…The molecular weight of the liver 6PGD from yellow catfish was 50.1 kDa by SDS-PAGE, similar to those in Dicentrarchus labrax L. liver [27], rat liver [13], rabbit mammary gland [25], human erythrocytes [26], sheep liver [28] and rat small intestine [10], lower than those in rat erythrocytes [5] and kidney [17], but higher than those in pig liver [8]. Generally speaking, dimer and/or tetramer is its active form for the enzyme, but dimer and tetramer possess different subunit mass.…”

Section: Kinetic Behaviour Of 6pgdmentioning

confidence: 93%

“…Ceyhan et al [10] suggested that the specific activity of 6PGD showed great variability. For example, the specific activities of 6PGD from mammals were reported to range between 0.41 and 22.6 U/mg protein [5,8,9,13,25,26].…”

Section: Kinetic Behaviour Of 6pgdmentioning

confidence: 99%

Purification and kinetic characteristics of hepatic 6-phosphogluconate dehydrogenase (6PGD) from yellow catfish Pelteobagrus fulvidraco

et al. 2015

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Objective: To purify and characterize hepatic 6-phosphogluconate dehydrogenase (6PGD) from yellow catfish and evaluate the effect of several metal ions (Cd 2+ , Cu 2+ and Al 3+) on the enzymatic activity in vitro. Methods: The 2', 5'-ADP Sepharose 4B chromatography is used as the purification procedure; SDS-PAGE is used to assay the 6PGD purity and determine the molecular mass of the subunit; 6PGD activity is determined following rates of the conversion of NADP + to NADPH at 340 nm. The optimal temperature, pH and ionic strength is determined; The substrates and product kinetic, effect of metal ions are studied. Results:The specific 6PGD activity is 5.75 U/mg, and its molecular weight is 50.1 kDa with a signal band. The optimum pH, temperature and ionic strength for the enzyme are pH 7.85, 60°C and 100 mM, respectively. The enzyme has the activation energy of 17.37 kcal/mol. 24, 2015] body weight daily. Each tank was provided with continuous aeration to maintain the dissolved oxygen level above saturation. Water in each tank was thoroughly replenished daily. Fecal matter was removed daily before feeding. We assure that the experiments performed on animals, animal care, and all protocols followed the ethical guidelines of Huazhong Agricultural University.During the experiment, the water quality parameters were measured in the morning twice a week. Dissolved oxygen was more than 5.3 mg l -1 , pH=6.9~8.5, total ammonia-nitrogen 0.04-0.064 mg l -1 , and water temperature 25±3 o C. SamplingAt the end of the 2-wk experiment, fish were starved for 24 h before sampling. Then they were killed by severing of the spinal cord. The liver was isolated immediately using sterile forceps in ice, quickly frozen in liquid nitrogen, and stored at -80 o C for subsequent analysis. Purification of liver 6PGD6PGD was purified according to the recently published protocols [17] with slight modification. The purification procedure consisted of 2', 5'-ADP-Sepharose 4B affinity chromatography. All the procedures were carried out at 4 o C.At first, the liver was homogenized in a glass-Teflon homogenizer with 3 volumes of buffer A (10 mM Tris-HCI buffer containing 5 mM 2-ME and 1 mM EDTA, pH 7.85) on the ice. The homogenate was centrifuged at 100,000 g for 60 min at 4°C. The supernatant was loaded onto 2'5'-ADP-Sepharose 4B column (1.6 cm×10 cm) preequilibrated with buffer A. The column was washed with the buffer A at the flow rate of 18 ml/h, by means of a peristaltic pump until the absorbance at 280 nm declined to 0.03 O.D. Then, the enzyme of G6PD was eluted with 10 mM Tris-HCl + 5 mM 2-ME + 1 mM EDTA + 0.2 mM NADP + , pH 7.85. After G6PD was washed, 6PGD was eluted using buffer A containing 1 mM NADP + , pH 7.85. The flow rates for washing and equilibration were reduced to 10.8 ml/h. Active 6PGD fractions were collected. Purification scheme of 6PGD from yellow catfish liver was shown in Table 1. The determination of 6PGD activityThe hepatic 6PGD activity of yellow catfish was determined at 25°C by following rates of the ...

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“…Parallel samples and reference proteins (fi-galactosidase, bovine serum albumin, ovalbumin and lysozyme) were co-electrophoresed. The duplicate samples and reference gels 16 were stained for protein with Coomassie Blue (Betts & Mayer, 1975) The time-dependent incorporation of mannose into microsomal fractions depended on the homogenization buffer. With a microsomal fraction prepared in phosphate buffer (Fig.…”

Section: Analysis Of Residualprotein Materialsmentioning

confidence: 99%

The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland

White¹

1978

Biochemical Journal

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1. A lactating rabbit mammary-gland microsomal system catalysed the incorporation of mannose from GDP-[U-14C]mannose into three endogenous acceptors, (i) polyprenyl phosphate mannose, (ii) lipid-linked oligosaccharide and (iii) protein. 2. Synthesis of polyprenyl phosphate mannose was stimulated by addition of dolichol phosphate to the incubation medium and was reversed by addition of GDP. The product had properties identical with those of authentic dolichol phosphate mannose. 3. The oligosaccharides derived from acid hydrolysis of the lipid-linked oligosaccharide fraction were of six, eight and nine to ten monosaccharide units, the octasaccharide being the major species formed. The oligosaccharide appeared to be attached to the lipid via a pyrophosphate bridge, since strong alkaline hydrolysis liberated an oligosaccharide phosphate. 4. Polyprenyl phosphate mannose served as a mannose donor to lipid-linked oligosaccharides and protein. When added as exogenous substrate it gave rise to a lipid-linked oligosaccharide of about six units. 5. Incorporation of radioactivity in protein was low, but polyacrylamide-gel electrophoresis of the protein fractions indicated that polypeptides of mol.wts. 115000, 75000 and 33000 were labelled.

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Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

Cited by 33 publications

References 15 publications

Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland

Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland

Purification and kinetic characteristics of hepatic 6-phosphogluconate dehydrogenase (6PGD) from yellow catfish Pelteobagrus fulvidraco

The formation of lipid-linked sugars by cell-free preparations of lactating rabbit mammary gland

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