Francis S. Markland

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.

Characterization and comparison of the estrogen and androgen receptors from the R-3327 rat prostatic adenocarcinoma.

Abstract The estrogen and androgen receptors found in the high-speed supernatant (cytosol) fraction of the Dunning R-3327 rat prostatic adenocarcinoma have been characterized and their properties compared. [ 3 H]-17β-Estradiol is bound to a macromolecule which sediments at 8.3 S after sucrose density gradient centrifugation in low salt buffer, whereas [ 3 H]-R1881 (methyltrienolone, a synthetic androgen) binds to a macromolecule sedimenting at 7.8 S. Competitive binding studies distinguish the two receptors and indicate that they are unique and distinct proteins. The dissociation constants for 17β-estradiol and R-1881 binding to their receptors are 8 × 10 −10 and 3 × 10 −9 , respectively. Both Scatchard plots are linear, suggesting a single class of high-affinity binding sites for each receptor. Steroid binding for both receptors was saturable and of low capacity. Estrogen receptor levels varied from barely detectable to 300 fmol/mg cytosol protein and androgen receptor levels ranged from barely detectable to 300 fmol/mg cytosol protein depending on tumor pathology. Tumors with large amounts of fibrous stroma had low receptor levels, whereas those tumors that were mostly epithelial in nature contained significantly higher levels of the receptors. Both the estrogen and androgen binding proteins satisfy criteria which clearly distinguish them from other (plasma) steroid binding proteins and classify them as true steroid hormone receptors.

Affinity-labelling corticoids I. Synthesis of 21-Chloroprogesterone, Deoxycorticosterone 21-(1-Imidazole) Carboxylate, 21-Deoxy-21-Chloro Dexamethasone, and Dexamethasone 21-Mesylate, 21-Bromoacetate, and 21-Iodoacetate

Abstract The efficient and unambiguous preparation of several C-21 substituted affinity-labelling corticoids are described. Included is an improved procedure for the preparation of 21-chloroprogesterone and the first reported synthesis of deoxycorticosterone 21-(1-imidazole) carboxylate. Dexamethasone derivatives prepared include separate and unambiguous synthesis of 21-deoxy-21-chlorodexamethasone and dexamethasone 21-mesylate, as well as the first reported synthesis of dexamethasone 21-bromoacetate and dexamethasone 21-iodoacetate.

Isolectric 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.

Comparison of two methods for proteolytic enzyme detection in snake venom

An acrylamide gel system containing fibrinogen was used to detect proteolytic enzymes in snake venoms. Proteolytic activity was observed as a clear area on a blue background after electrophoresis and overnight incubation in Tris buffer, prior to staining with Coomassie blue. Venoms from eastern and western diamondback and west coast Mexican rattlesnakes, Crotalus adamanteus, C. atrox and C. basiliscus basiliscus, respectively, and southern copperhead, Agkistrodon contortrix contortrix, were analyzed at the level of 1 mg of venom. The effect of the serine proteinase inhibitor diisopropylfluorophosphate (DFP) and the metalloproteinase inhibitors tetraethylenepentamine (TEP) or EDTA on fibrinogen and normal gel profiles were evaluated. Normal gels (without fibrinogen) were Coomassie stained to visualize migration of 250 micrograms of venom proteins on the gels. Several proteolytic enzymes detected in C. atrox and C. b. basiliscus venoms were inhibited by TEP, whereas DFP had no effect on activity. The fibrinogen gels detected no protease activity in C. adamanteus venom, although it is known from other studies that there are several proteolytic enzymes in this venom. Several proteases were detected in A. c. contortrix venom, one of which was inhibited by TEP. By comparison, proteolytic activity in 5-10 micrograms of all venoms was readily detected using the mammalian plasma kallikrein specific chromogenic substrate, S2302 (H-D-Pro-Phe-Arg-p-nitroanilide). The fibrinogen gel method does not appear to have the specificity nor the sensitivity of the recently developed chromogenic substrates for detection of proteolytic enzymes in snake venom.

Competitive binding assay for glucocorticoids. Influence of experimental conditions on measurement of the affinity of competitive steroids for the receptor.

Abstract The affinity of steroids for a receptor is frequently determined by the competitive binding assay. This assay compares, under equilibrium conditions, the amount of a specific radioactive steroid bound to the receptor in the presence and absence of various concentrations of the radioinert steroid to be studied. The results are expressed as relative binding affinities, which relate the binding ability of the radioinert competitors to that of the radioactive steroid. If is often assumed that the time needed for the radioactive steroid to equilibrate with the receptor is the same both in the presence and absence of radioinert competitors. In this report, using molybdate-stabilized glucocorticoid receptor from goat lactating mammary gland, we demonstrate that the time required to reach equilibrium when both the radioactive and radioinert steroid are present is much longer than when only the radioactive steroid is present. The apparent relative binding affinity varies continuously as the incubation time is extended and the rate of change depends not only on the temperature of the assay but also (and more importantly) on the degree of difference in absolute affinity between the specific radioactive steroid and the unlabelled competitor being compared. Therefore, in the competitive binding assay the conditions necessary to reach equilibrium are influenced by experimental variations and may be different for each steroid tested. Thus, large errors are possible when comparing results obtained using different temperatures, lengths of incubation or substances which may influence steroid binding kinetics. We believe that kinetic analysis is preferable to equilibrium analysis whenever a precise study of the interaction of radioinert steroids with a receptor is needed.

PREFACE: SNAKE VENOMS and HEMOSTASIS

In this special series of Toxin Reviews I have invited leading authorities who have played important roles in the advancement of their fields of toxinology and hemostasis to contribute chapters to this series. I would like to apologize to all of those investigators who have made equally important contributions to this field, but because of space limitations could not be included in this special series. In preparing these chapters, the authors were asked to present a brief review of the field as well as provide an update on their own research activity. This special series contains thirteen chapters covering many areas of research interest and clinical studies related to the effects of snake venoms on the hemostatic system. In the first Chapter Dr. Neville A. Marsh opens the series with a brief, but very interesting, historical overview of snake venoms that affect hemostasis. As so elegantly pointed out by Marsh: “These toxins are diverse both in form and function: whilst once the source of prolific basic research, snake venom toxins now afford many opportunities for both clinical and laboratory use.” The first chapter is followed by four chapters describing the effect of snake venoms on different factors in the coagulation cascade. In Chapter 2, Drs. Gerry A. F. Nicolaes and Jan Rosing describe the activation and inactivation of blood coagulation factor V by snake venom proteinases. In Chapter 3, Drs. Jüri Siigur and Ene Siigur discuss the structure and function of Factor X activating proteinases from snake venoms. In Chapter 4, Dr. Manjunatha Kini and Md. Abu Reza discuss structural characteristics and evolution of the venom prothrombin activators and blood coagulation factors from Australian snakes. In Chapter 5, Drs. Helena C. Castro and Carlos Rangel Rodrigues focus on the current status of snake venom defibrinogenating (thrombin-like) enzymes with particular reference to those enzymes whose structures have been studied in detail. In putting together this series, I attempted to include several chapters on the diagnostic use and clinical applications of snake venom proteins and the next two chapters are specifically directed towards this goal. In Chapter 6, Drs. David E. Levy and Gregory J. del Zoppo discuss