Detection of the membrane inhibitor of reactive lysis (CD59) in diseased neurons of Alzheimer brain.

The membrane inhibitor of reactive lysis (MIRL) protects host cells from complement-mediated lysis. It was detected immunohistochemically in tangled neurons and dystrophic neurites of Alzheimer disease (AD) tissue in a pattern highly similar to that observed for the membrane attack complex of complement, C5b-9. MIRL was also detected in cultured IMR-32 neuroblastoma cells. The mRNA for MIRL was detected in RNA extracts of both AD and normal brain. These data provide the first evidence of brain neuronal expression of MIRL and its upregulation in neurons exposed to complement attack. They are consistent with the previously advanced hypothesis that complement-mediated neuronal injury may play a role in AD.

Immunological properties of the fibrinolytic enzyme (fibrolase) from southern copperhead (Agkistrodon contortrix contortrix) venom and its purification by immunoaffinity chromatography.

An antibody to the fibrinolytic enzyme in southern copperhead venom was produced by immunizing rabbits with chromatographically purified enzyme. The antibody was purified from rabbit blood by ammonium sulfate fractionation and protein-A affinity chromatography. The purified antibody reacted only with the fibrinolytic enzyme in southern copperhead venom as demonstrated by immunodiffusion and immunoelectrophoresis. Western immunoblotting revealed that several snake venoms, including Agkistrodon piscivorus conanti, Crotalus atrox, Crotalus basiliscus basiliscus, and Bothrops asper, cross-reacted with the antibody to varying degrees. However, Deinagkistrodon acutus showed no cross-reaction. Immobilized antibody has been used, in combination with molecular sieve chromatography, to purify the fibrinolytic enzyme from southern copperhead venom. In this two-step purification procedure, the enzyme was purified in good yield within two days. The specific activity of the enzyme purified by the immunoaffinity chromatography procedure is comparable with that of enzyme purified by a four-step chromatographic procedure. The mol. wt of the purified enzyme is approximately 23,000-24,000 as determined by SDS-PAGE. Interestingly, the enzyme purified by this two-step immunoaffinity chromatography procedure possesses virtually no hemorrhagic activity.

Isolation by preparative isoelectric focusing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead).

Fibrolase, a blood clot-lysing enzyme, was isolated from the venom of the snake Agkistrodon contortrix contortrix using preparative scale isoelectric focusing in the recycling isoelectric focusing (RIEF) apparatus. Two sequential purifications, beginning with 1.0 g of whole, dried venom, were employed. A pH 6-8 range gradient effected the first separation. While 100% of the enzyme was recovered in three fractions, 43% (one fraction) had 70% purity. The second run was a refractionation of three, pooled fractions from the first run, in a 0.7 pH range gradient. Of the fibrolase in the venom, 63% was recovered in four fractions. One of these represented 29% of venom fibrolase, with 97% purity. Gel filtration chromatography removed most of the remaining, higher molecular weight contaminants of the RIEF-purified enzyme.

Isoelectric focusing in immobilized pH gradients of a snake venom fibrinolytic enzyme.

A fibrinolytic enzyme with a molecular weight between 23,000 and 25,000 Da has been purified from southern copperhead snake venom. Immobilized pH gradient isoelectric focusing with an ultranarrow pH interval (pH 6.65-6.95) resolved two isoforms of the fibrinolytic enzyme that were not resolved by standard isoelectric focusing. Attempts at purification of the individual isoenzymes by semi-preparative scale IPG and elution of enzyme by macerating the gel yielded only 20-40% recovery of activity. In attempts to improve recovery, a semi-preparative IPG canal-isoelectric focusing technique has been utilized.

Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix).

A fibrinolytic enzyme present in Agkistrodon contortrix contortrix (southern copperhead) venom has been purified by combination of CM-cellulose chromatography, molecular sieve chromatography on Sephadex G-100, p-aminobenzamidine-agarose affinity chromatography, and DEAE-cellulose chromatography. The enzyme, fibrolase, has a molecular weight of 23,000-24,000 and an isoelectric point of pH 6.8. It is composed of approximately 200 amino acids, possesses a blocked NH2-terminus and contains little or no carbohydrate. The enzyme shows no activity against a series of chromogenic p-nitroanilide substrates and is not inhibited by diisopropylfluorophosphate, soybean trypsin inhibitor, Trasylol, or p-chloromercuribenzoate. However, the enzyme is a metalloproteinase since it is inhibited by EDTA, o-phenanthroline and tetraethylenepentamine (a specific zinc chelator). Metal analysis revealed 1 mol of zinc/mol of protein. Study of cleavage site preference of the fibrinolytic enzyme using the oxidized B chain of insulin revealed that specificity is similar to other snake venom metalloproteinases with cleavage primarily directed to an X-Leu bond. Interestingly, unlike some other venom fibrinolytic metalloproteinases, fibrolase exhibits little if any hemorrhagic activity. The enzyme exhibits direct fibrinolytic activity and does not activate plasminogen. In vitro studies revealed that fibrolase dissolves clots made either from purified fibrinogen or from whole blood.