Comparative study on glucose-6-phosphate dehydrogenase from rabbit tissues

The activity of glucose-6-phosphate dehydrogenase (G6PD) was measured in bone marrow, spleen, lung, liver, kidney, adipose tissue, brain, heart, muscle, and in the erythroid cell line of rabbit. In tissues, the activity ranged from 6.87 to 0.09 U/g wet tissue, found in bone marrow and muscles, respectively, whereas in the erythroid cell line it ranged from 14.3 to 2.4 U/g cells for erythroblasts and erythrocytes, respectively. The electrophoretic patterns of the tissue crude extracts showed an identical set of three activity bands, and the immunotitration curves obtained with rat antirabbit erythrocyte G6PD antibodies shared the same equivalence point. The enzyme, purified to homogeneity from different tissues, showed no significant differences among the Km values for NADP and G6P. The results give a picture of the variability of the G6PD activity in rabbit tissues and suggest the presence of the same enzyme molecule in each tissue.     

Hepatic hematopoiesis in phenylhydrazine-induced hemolytic anemia

Ten adult rabbits were divided into two groups: the control rabbits, which received subcutaneous injections of 0.9% NaCl in three days; the experimental animals which received 3 mg/Kg body weight of phenylhydrazine (PHZ) subcutaneously also in three days. On the 8th day from the initial treatment the control and experimental animals were sacrificed, blood was collected to determine hematological parameters and livers were cut into small pieces. Sections were prepared by pressing the pieces onto slides which were stained with the Giemsa stain. The hematocrit and the reticulocytosis of experimental animals were 25 + 3%, and 70 + 5% respectively. In the liver sections of the PHZ treated animals we found a very rich population of immature erythroblasts. In fact proerythroblasts and basophilic erythroblasts were 19%, polychromatic and orthochromatic erythroblasts were 22% and 13% respectively. On the contrary, these cells were absent in the control livers. The lymphocyte and lymphoblast population, on the other hand, was very rich in control animals with a value of 38.8% compared to 1.62% in the anemic animals. The results clearly indicate the hematopoietic function of the liver in the anemic animals although the low percentage of orthochromatic erythroblasts with respect to their precursors suggests the ineffectiveness of the process.     

One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography

Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to -tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.     

Total extract of Beta vulgaris var. cicla seeds versus its purified phenolic components: antioxidant activities and antiproliferative effects against colon cancer cells

Introduction – Beta vulgaris var. cicla (BV) leaves contain chemopreventive compounds that have been investigated for new drug discovery. These compounds belong to the family of the apigenin‐glycosides. Since the leaves are seasonal products containing high percentages of water, they are easily degradable during storage in fresh conditions. To be stored they require a drying process, consuming time and a large amount of energy. The extraction of apigenin‐glycosides may also be conveniently performed from BV seeds, which represent a stable and year‐long available biomass. Objectives – The present report was undertaken to find a strategy of purification of bioactive flavonoids from BV seeds and test their ability to inhibit proliferation both on human colon cancer (RKO) cells and normal human fibroblasts (HF). Materials and methods – The ethyl‐acetate extract of BV seeds was fractionated on a Sephadex LH 20 column. A fraction of this extract, labeled as P4, exploited a marked antiproliferative activity on RKO cells. The components of P4 were purified on an RP₁₈ column chromatography and identified by HPLC‐ESI‐MS as 2,4,5‐trihydroxybenzaldehyde, 2,5‐dihydroxybenzaldehyde, vanillic acid, xylosylvitexin, glucopyranosyl‐glucopyrasyl‐rhamnetin and glucopyranosyl‐xylosyl‐rhamnetin. All of them were tested for cytostatic and cytotoxic activity on RKO and HF cells. Results – Xylosylvitexin exhibited the strongest antiproliferative activity on RKO cells, together with an enhancement of the apoptosis, an increase of cells in the G₁ phase and a reduction of cells in the S phase; on the contrary, the proliferation of HF was significantly stimulated. Conclusion – Xylosylvitexin is the main and more efficient chemopreventive compound in BV seeds, but the natural cocktail of molecules, represented by P4 fraction, showed a better compromise between the antiproliferative activity on RKO cells and the enhancement of HF proliferation. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.     

Effect of ATP on glucose-6-phosphate dehydrogenase during erythroblast maturation in the rabbit

The glucose-6-phosphate dehydrogenase (G6PD) activity of erythroblasts, separated at different advancing stages of development, shows a marked decline of activity. A proteolytic mechanism, strictly controlled, is likely responsible of this decay, since a sufficient level of enzyme activity still remains in the circulating erythrocyte. In this report we suggest a model that could explain what triggers the mechanism of proteolytic degradation. HPLC analysis of the nucleotide content of erythroblasts and reticulocytes, showed a marked decline of adenine and pyridine nucleotides and of their catabolic products during the cell development. From thermostability tests, at fixed temperature, we have seen that ATP and NADP only, significantly protected the enzyme activity. In this light, we incubated 10 min at increasing temperatures, with and without ATP or NADP lysates of erythroblasts, separated at different stage of development and of reticulocytes. In the absence of nucleotides, we determined for all fractions a T degree break at 42 degrees C. In the presence of NADP all fractions were stabilized with no break point in the range 37-50 degrees C. On the contrary, the presence of ATP caused a progressive shift of the T degrees C break from the most immature erythroblasts (T degree break at 46 degrees C) to the reticulocytes (T degree break at 42 degrees C). Since ATP did not show any protective effect on the reticulocyte enzyme, we hypothesize the presence in these cells of a structurally modified G6PD. Furthermore, these data support our belief that the marked decline of ATP during cellular development, may represent the element responsible for the enzyme modification.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Postnatal Expression of Glucose-6-Phosphate Dehydrogenase in Different Brain Areas

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in five brain areas of rats aged 5 to 90 days. The areas studied were: the olfactory bulb (OB), cortex, hippocampus, striatum and septum. The G6PD activity increased more than 2-fold from 5 to 90 days in the OB, while it was almost constant in the other areas. At every stage of development, the G6PD activity was significantly higher in the OB than in the other areas. The G6PD pattern was compared with 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR); glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) in order to find synergistic interactions among activities of these enzymes during development. Over the considered period, the activity of 6PGD increased significantly in the OB, while no significant difference in activity was detected in the other areas. GR increased significantly and progressively at each developmental stage in all areas. GPX showed a progressive increase in the OB, while in other areas a significant increase was detected at 90 days only. CAT and SOD showed a different and independent pattern which differred from the G6PD pattern. CAT showed the highest level of activity at 5 days then progressively decreased or was constant until 90 days; SOD had the highest value at 5 days, than it decreased at 10 days and increased from 10 to 90 days. In all areas, G6PD activity showed three electrophoretic bands, whose relative activity changed with development. At histochemical level, we found a marked G6PD activity in the periglomerular zone of the OB, which increased with age, while other areas showed a homogeneous staining. The present results demonstrate that G6PD activity increases in the OB during the developmental stages and there is a coordinated simultaneous activation of 6PGD, GPX and GR. It is likely that this enzyme induction increases the antioxidant defense of periglomerular cells that are subject to a rapid renewal and thus much more exposed to oxidant stress.     

Posttranslational regulation of glucose-6-phosphate dehydrogenase activity in tongue epithelium.

Expression of glucose-6-phosphate dehydrogenase (G6PD) activity is high in tongue epithelium, but its exact function is still unknown. It may be related either to the high proliferation rate of this tissue or to protection against oxidative stress. To elucidate its exact role, we localized quantitatively G6PD activity, protein and mRNA using image analysis in tongue epithelium of rat and rabbit, two species with different diets. Distribution patterns of G6PD activity were largely similar in rat and rabbit but the activities were twofold lower in rabbit. Activity was two to three times higher in upper cell layers of epithelium than in basal cell layers, whereas basal layers, where proliferation takes place, contained twice as much G6PD protein and 40% more mRNA than upper layers. Our findings show that G6PD is synthetized mainly in basal cell layers of tongue epithelium and that it is posttranslationally activated when cells move to upper layers. Therefore, we conclude that the major function of G6PD activity in tongue epithelium is the formation of NADPH for protection against oxidative stress and that diet affects enzyme expression in this tissue.     

Progression of the peripheral blood profile in phenylhydrazine-induced hemolytic anemia

Male rabbits were made anemic by subcutaneous injections of 3 mg/Kg body weight of phenylhydrazine (PHZ) for the duration of three days. Blood samples were collected prior to the experiment, on the 2nd and 5th day from the end of the treatment. Hematocrit, reticulocyte count, MCV, osmotic resistance, RBC volume distribution curve and morphological analysis were performed on each sample in order to obtain a picture of the progressive changes of the parameters following the PHZ administration. Hematocrit values changed from 49 to 27%, whereas the reticulocytosis increased from 0.6 to 73%. A significant elevation of MCV was detected and the cell volume distribution curve, which presented the typical bell-shape profile in the normal animals became bimodal on the 5th day. The osmotic resistance of RBC of anemic animals, showed a marked deviation from the normal pattern. In fact initial haemolysis started at 0.75-0.70% NaCl concentration in the 5th day samples, while it was between 0.55-0.50% before the PHZ treatment. Finally, morphological analysis revealed a progressive increase of RBC with Heinz bodies, of macro-megalocytes and of immature erythroblasts thus indicating that the cell population, produced during recovery from PHZ induced anemia, is widely heterogeneous.