Daboxin P,a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A₂ activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca⁺² binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A₂ enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity.     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Aging shifts mitochondrial dynamics toward fission to promote germline stem cell loss

Changes in mitochondrial dynamics (fusion and fission) are known to occur during stem cell differentiation; however, the role of this phenomenon in tissue aging remains unclear. Here, we report that mitochondrial dynamics are shifted toward fission during aging of Drosophila ovarian germline stem cells (GSCs), and this shift contributes to aging‐related GSC loss. We found that as GSCs age, mitochondrial fragmentation and expression of the mitochondrial fission regulator, Dynamin‐related protein (Drp1), are both increased, while mitochondrial membrane potential is reduced. Moreover, preventing mitochondrial fusion in GSCs results in highly fragmented depolarized mitochondria, decreased BMP stemness signaling, impaired fatty acid metabolism, and GSC loss. Conversely, forcing mitochondrial elongation promotes GSC attachment to the niche. Importantly, maintenance of aging GSCs can be enhanced by suppressing Drp1 expression to prevent mitochondrial fission or treating with rapamycin, which is known to promote autophagy via TOR inhibition. Overall, our results show that mitochondrial dynamics are altered during physiological aging, affecting stem cell homeostasis via coordinated changes in stemness signaling, niche contact, and cellular metabolism. Such effects may also be highly relevant to other stem cell types and aging‐induced tissue degeneration.