Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 æmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 æmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18 percent, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Partial purification and some physicochemical properties of phospholipases A2 from the venom of the bushmaster snake (Lachesis muta)

Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05 per cent of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phospholipase A2 activity and their inhibitory effect on collagen-induced platelet aggregation

Purification, physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom

Snake venoms usually contain multiple molecular forms of phospholipase A2 enzymes (phosphatide acyl hydrolase, E.C. 3.1.1.4; PLA2). Phospholipases A2 induce a wide range of pharmacological effects which may depend or not on the hydrolysis of phospholipids. In this study, a PLA2 from Bothrops jararaca venom was purified to homogeneity by gel filtration on a Sephacryl S-200 column, followed by FPLC reverse-phase chromatography on a Pep-RPC HR 5/5 column (yield 1.63 per cent of venom protein). The PLA2 activity of the fractions was determined by indirect hemolysis using hen's egg yolk lecithin as substrate. The enzyme is an acidic protein with PI 4.5 and an apparent molecular weight of 14,200, as estimated by gel filtration on a Superose 12 FPLC column. Similar properties have been described for PLA2 from other snake venoms. The N-terminal-sequence of the purified protein was NLMQFETMIMXXAGQ. These partial sequence data show a high degree of homology between the B. jararaca PLA2 and the enzymes from other snake venoms as well as bovine pancreatic PLA2

Biochemical and pharmacological screening of snake (Bothrops) venoms: characterization of components acting on blood coagulation and platelet aggregation

Six venoms from snakes of the genus Bothrops were tested for coagulation, platelet aggregation and phospholipase A2 (PLA2) activity. Almost all showed pro-coagulant and PLA2 activities while pro-aggregating properties were found only for some venoms. Bothrops jararaca venom showed different protein peaks associated with these activities. The pro-aggregating activity was inhibited by EDTA, leupeptin and mepacrine while the PLA2 activity was blocked by p-bromophenacyl bromide and 2-mercaptoethanol. Venom screening tests for clotting and platelet aggregation may represent a valuable tool for snake taxonomy and for monitoring the quality of antisera