Effect of Metabolism of Carbohydrate in Mammalian Tissues and Tumors on The Level of Enzymes Involved in Direct Oxidative Pathway

Introduction: Literature review has revealed that the distribution of the enzymes of 6-phospo-gluconate dehydrogenase activities of some acetone derived tissues in animal tissue have so far not been investigated systematically. This leaves a gap for further investigation to explore the subject matter deeply. Method: Barium salts of D-Glucose 6-phosphate (0 6-P), 6-phosphogluconate (6-PO) and D-ribose 5-phosphate (R 5-P) are available and were used in our study. (TNP) triphosphopyridine was prepared and analyzed; its composition was 75% TNP without (DNP) diphosphopyridine nucleotide. Ice-cold isotone KCL (0-15M-KCL with 8ml, 0-02M-KHCO3) was disintegrated in 09 parts. It is done in a potter glass homogenizer or in a Nelco homogenizer. This is followed by centrifuging and dialysis of the supernatants. Heparinized blood 10ml was used for erythrocyte hemolysis, which was diluted with 10ml of water. 01 part of haemolysate was treated with 9 parts of isotonic KCI. Spectroscope is used to determine the dehydrogenase activity of dialyzed tissue. The method followed was of Glock and McLean. Study Design: Quantitative, cross sectional study. Settings: Institute of Biochemistry, Gulab Devi Educational Complex, Lahore Duration: 01 Year i.e. 1st July 2020 to 30th June 2021. Results: Enzymes activities of 6-PG dehydrogenase and Gluco-6- phospo dehydrogenase mentioned in table 1&2 in normal mammalian tissue and mammary glands. The results obtained on the tumour cells are given in table 3. These Values are within the limits in normal tissues whereas it becomes on higher side in lymphomas and sarcomas. Conclusion: This study shows some limitation that the maximum enzymic activities are determined, whereas in the intact cell other regulatory factors probably limit or control the activity of this pathway. Keywords: Gluco-6- phospodehydrogenase, 6-PG dehydrogenase, oxidative pathway, Ribose 5-Pentose, mammalian tissue

Ferritin immunoelectron microscopic localization of 5'-nucleotidase on rat liver cell surface.

Rat livers were prefixed by perfusion with 0.6% glutaraldehyde and briefly homogenized with a Teflon-glass homogenizer. The prefixed cells isolated by low-speed centrifugation in high yield effectively preserved the original polygonal shape and polarity. These cells were incubated with ferritin-antibody conjugates monospecific for rat liver 5'-nucleotidase, and the localization of the enzymes on the surface of hepatocytes and endothelial cells was quantitatively investigated. It was revealed that the surface density of 5'-nucleotidase is much higher on the bile canalicular surface than on the sinusoidal surface and only a few ferritin particles were detected on the lateral surface. On the bile canalicular surface ferritin particles were almost exclusively found on the microvilli in larger clusters. Similar distribution was also observed on the sinusoidal surface but the size of cluster was much smaller. On both surfaces many fewer ferritin particles were found on the intermicrovillar region, including the coated pits region, than on the microvillar region. Ferritin particles were also found on the endothelial cell surface.

PREPARATION AND PROPERTIES OF A MITOCHONDRIAL FRACTION FROM WHEAT STEM RUST UREDOSPORES

Ungerminated uredospores of Puccinia graminis f. sp. tritici Erikss. & Henn. race 15B were disrupted in a buffered sucrose serum albumin solution, using a "Teflon" pestle homogenizer and glass beads 80 μ in diameter. A particulate fraction was sedimented between 2000 and 30,000 × g and its oxidation of Krebs' cycle acids measured manometrically. Endogenous respiration of washed preparations was low or negligible. Oxygen uptake was observed with succinate (40 to 60 μl/hour mg protein), α-ketoglutarate, malate, citrate, and isocitrate (10 to 30 μl/hour mg protein) but not with fumarate or pyruvate. Succinate oxidation was sensitive to heat, cyanide, and malonate.Aspects of the extraction procedure were examined for effects on yield and activity. Serum albumin in the grinding medium, very low grinding speed, and short grinding time favored high activity per mg protein. Glass beads increased the yield with the "Teflon" pestle while an all-glass homogenizer gave preparations of low activity.Oxidatively active particle suspensions reduced Janus green B and were shown by electron microscopy to consist of vesicles and some mitochondria.

A METHOD FOR ISOLATING INTACT MITOCHONDRIA AND NUCLEI FROM THE SAME HOMOGENATE, AND THE INFLUENCE OF MITOCHONDRIAL DESTRUCTION ON THE PROPERTIES OF CELL NUCLEI

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0–6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl2 is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The α-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0–6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.