Structural diversity of trypsin from different mosquito species feeding on vertebrate blood

Mosquito trypsin was purified using a combination of ion exchange and affinity chromatography with the ligand soybean trypsin inhibitor. Three Aedes and three Anopheles species were tested, all of which are specialized in the digestion of vertebrate blood. Amino-terminal sequences of HPLC-purified trypsins from Aedes aegypti and Anopheles quadrimaculatus revealed homologies of 30-40% with vertebrate and other invertebrate proteases previously identified as serine-proteases. The purified mosquito trypsins have molecular masses between 25 kDa and 36 kDa, as determined by denaturing polyacrylamide electrophoresis, and are heterogeneous in size and number in the various species. The number of SDS-bands varies between 3 and 6 in Aedes and between 1 and 3 in Anopheles. The specific activities, determined with the substrate TAME, range from 240 U/mg in Aedes aegypti to 1065 U/mg in Anopheles quadrimaculatus. All mosquito trypsins tested have acidic isoelectric points between pH 3.5 and pH 5.4. No alkaline proteases were detected. Polyclonal antisera against Aedes aegypti and Anopheles albimanus trypsin do not cross-react with bovine trypsin. Cross-reactivity of the two sera with trypsin from six mosquito species suggests the presence of at least 2 enzyme families.     

Model of the interaction between trypsin and alpha 2-macroglobulin of porcine serum]

A comparative study of the dissociation into subunits of Porcine alpha2 M, either native or bound to trypsin (Tn), has been carried out in order to determine the modifications of the alpha2 M structure due to the formation of the Tn-alpha2 M complex. Analytical ultra-centrifugation at pH 3.5 shows that the dissociation is smaller when alpha 2 M is bound to trypsin. Electrophoresis in 4% polyacrylamide gels, in presence of 0.1% SDS, of alpha2 M and Tn-alpha2 M incubated in 1% SDS leads to the same conclusion; the enzyme must stabilize the quaternay structure of alpha2 M. In presence of SDS + beta-mercaptoethanol, only a molecular weight (M.W.) 200,000 band is revealed in electrophoresis pattern of native alpha2 M. In the case of reduced Tn-alpha2 M, some other bands of M.W. 100,000, 50,000, 30,000 appear. When trypsin is inactivated by TLCK 100,000 M.W. band is present, accompanied by the 200,000 M.W. band whose intensity is function of the alpha2 M concentration. The 100,000 M.W. band appears therefore characteristic of the formation of the complex which must imply a proteolytic cleavage in the middle of the 100,000 polypeptidic chain of alpha2 M. A model of the complex is proposed in which the enzyme forms a proteic bridge between the two halves of the alpha2 M molecule.     

Specificity of action of phospholipases of rat intestinal mucosa]

The total homogenate of rat intestine is devoid of C or D phospholipase activity. At pH 6,5 in the presence of phosphatidylcholines, it exhibits a B phospholipase activity and in the presence of exogen lysophosphatidylcholines an A1 lysophospholipase activity. At pH 8,5 the intestinal mucous membrane of a rat shows an isolated A2 phospholipase activity.     

Possible regulation of membrane-associated cyclic AMP phosphodiesterase in rat cerebral cortex by lipids.

Cyclic AMP phosphodiesterase (PDE) in membrane fraction from rat cerebral cortex was activated by Triton X-100, and treatment at alkaline pH and with phospholipase C. These results suggest that membrane PDE exists in a latent form and is influenced by microenvironmental changes within the membrane. Furthermore, the PDE, unlike soluble enzyme, is not influenced by a protein activator and Ca++.     

Structural diversity of trypsin from different mosquito species feeding on vertebrate blood

Summary Mosquito trypsin was purified using a combination of ion exchange and affinity chromatography with the ligand soybean trypsin inhibitor. ThreeAedes and threeAnopheles species were tested, all of which are specialized in the digestion of vertebrate blood. Amino-terminal sequences of HPLC-purified trypsins fromAedes aegypti andAnopheles quadrimaculatus revealed homologies of 30–40% with vertebrate and other invertebrate proteases previously identified as serine-proteases. The purified mosquito trypsins have molecular masses between 25 kDa and 36 kDa, as determined by denaturing polyacrylamide electrophoresis, and are heterogeneous in size and number in the various species. The number of SDS-bands varies between 3 and 6 inAedes and between 1 and 3 inAnopheles. The specific activities, determined with the substrate TAME, range from 240 U/mg inAedes aegypti to 1065 U/mg inAnopheles quadrimaculatus. All mosquito trypsins tested have acidic isoelectric points between pH 3.5 and pH 5.4. No alkaline proteases were detected. Polyclonal antisera againstAedes aegypti andAnopheles albimanus trypsin do not cross-react with bovine trypsin. Cross-reactivity of the two sera with trypsin from six mosquito species suggests the presence of at least 2 enzyme families.     

Possible regulation of membrane-associated cyclic AMP phosphodiesterase in rat cerebral cortex by lipids

Summary Cyclic AMP phosphodiesterase (PDE) in membrane fraction from rat cerebral cortex was activated by Triton X-100, and treatment at alkaline pH and with phospholipase C. These results suggest that membrane PDE exists in a latent form and is influenced by microenvironmental changes within the membrane. Furthermore, the PDE, unlike soluble enzyme, is not influenced by a protein activator and Ca⁺⁺.     

Effect of starvation on enzymes related to lipid metabolism in guinea-pig lungs

The changes in activities of acetyl CoA carboxylase, microsomal fatty acid elongation enzyme, choline phosphotransferase, triglyceride lipase, phospholipase A1 and phospholipase A2 were followed in guinea-pig lungs at 24, 48 and 72 h after food deprivation. Triglyceride lipase was elevated and phospholipase A1 and phospholipase A2 were unaffected, while the other activities decreased. The significance of these findings in relation to food deprivation is discussed.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH+4, increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, respectively.     

Trypsin activity in the midgut ofSarcophaga ruficornis andMusca domestica (Diptera: Insecta)

Summary The pH and temperature for the optimum activity of trypsin from the midgut ofSarcophaga ruficornis andMusca domestica was 7.5 and 8.0 respectively and 50°C. The enzymic activity increased with the increase in incubation period and enzyme concentration.Thanks are due to Prof.R. Rakshpal for guidance.     

The polyphosphate synthetase of Saccharomyces cerevisiae]

The polyphosphate-synthetase, isolated from a homogenate of phosphate starved cells, catalyses the synthesis of linear polyphosphates from orthophosphate. It is localized in the membrane fraction which deposits between 400 and 1000 X g; its optimal pH is 7.1; its KM toward orthophosphate is 4.0 X 10(-4) M; ATP stimulates the reaction. The enzyme synthezises especially polyphosphates with short chain length.     

Regulation and electric excitation of enzyme membranes in vitro]

Enzyme membranes can be activated or inhibited by applying continuous or alternating electrical fields. The field can modify the transport or reaction term of the transport-reaction by action on the displacement of charged species including those giving pH effects or inducing volume flows. A first experimental example is given: the progressive supression of the inhibition of hexokinase by the product when increasing alternating fields are applied. In the same way the apparent optimal pH approaches that of the soluble enzyme. In addition to its theoretical and practical implications electrical regulation can lead to the monitoring of enzyme reaction-driven mechanochemical fibers.     

Basic phospholipase of Naja nigricollis venom]

It is confirmed that N. nigricollis venom contains several phospholipases one of these is a basic phospholipase A. This enzyme is toxic for mice when injected intravenously. In vitro it reacts on egg yolk lecithin producing lysolecithin and prevents the phenomenon of blood clotting. An immunological identity has been established between this basic phospholipase and two acidic phospholipases present in the same venom.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

DOCA administration increases renal phospholipase activity in the rat

Summary The phospholipase activity of renal tissue has been evaluated in controls and in DOCA treated rats. DOCA treated animal showed a higher than normal enzyme activity. Since a phospholipase is the key step in prostaglandin biosynthesis, it is suggested that the increased prostaglandin release promoted by mineraloactive steroids is mediated by an activation of this key enzyme.This work has been partly supported by a grant from Consiglio Nazionale delle Ricerche.To whom reprint requests should be addressed.     

Formation of trypsin inhibitors during alkaline treatment of proteins

Casein is submitted to a severe alkaline treatment (NaOH 0,2 or 0,5 N, 1 hr., 80 degrees C). The hydrolysis by pancreatic enzymes (trypsin or chymotrypsin) is reduced in vitro and, in the case of the more severe treatment, stopped. After an extended (24 hrs.) trypsin and pronase hydrolysis, it is shown, by affinity chromatography, that peptides, which are not hydrolysable, can bind to trypsin and inactivate this enzyme in vitro.     

Serienbestimmung der Phospholipase-A-Aktivität in chromatographischen Fraktionen

Summary A simple assay is described for phospholipase A in chromatographic fractions. 0.01 ml of the fraction is added to 1 ml of a 1% solution of purified lecithin in ether (100 vol) — picoline (10 vol) — 4.5 mmol CaCl₂ (1 vol). The presence of phospholipase A is indicated by the appearance of a turbidity due to precipitation of lysolecithin. The lag period until precipitation occurs gives an indication of the relative concentration of enzyme present.     

Abtrennung des anaphylatoxinbildenden Prinzips aus Cobragift von anderen Giftkomponenten

Summary Cobra venom contains an anaphylatoxin-forming principle. This component has been purified by gel filtration and ion exchange chromatography. It has been obtained free from proteolytic or hemolytic activity as well as from phospholipase A. It seems to be an enzyme that splits the anaphylatoxin from its inactive precursor.     

Malignant hyperthermia: molecular defects in membrane permeability

Malignant hyperthermia (MH), a genetically inherited disorder of skeletal muscle, is due to molecular defect in membrane permeability. The alteration in membrane permeability is suggested to be due to enhanced phospholipase A2 activity which is responsible for the increased level in sarcoplasmic Ca2+. The excess Ca2+ is responsible for muscle hyper-rigidity and enhanced rate of glycolysis, resulting in a rapid rate of lactic acid production and a low pH in MH muscle.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Irreversible inactivation of yeast glucose-6-P dehydrogenase by penicillin G

Summary Yeast glucose-6-P dehydrogenase is irreversibly inactivated by penicillin G. Kinetic data show that 1 molecule of penicillin G reacts with each active unit when the enzyme is inactivated The rate of inactivation increases greatly with increasing pH. This irreversible inactivation by penicillin G is largely prevented by pyridoxal-P, a reversible inactivator of this enzyme. Prior treatment of penicillin G with penicillinase totally abolishes its ability to inactivate the enzyme.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resouces, NIH (USA).