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Kallikrein-like enzyme from the venom of Crotalus basiliscus basiliscus (Serpentes: Crotalidae).

Abstract. -- A kallikrein-like enzyme was isolated from the venom of Crotalus basiliscus basiliscus (Mexican west coast rattlesnake) and assayed for several physiological properties. Isolation of the enzyme was accomplished with the use of Sephadex G-75, DEAE (diethylaminoethyl) Sephadex A-50, QAE (diethyl [2-hydroxypropyl] aminoethyl) Sephadex A-50 and HPLC Aquapore CX-300 (carboxymethyl) chromatography columns. Fractions were assayed for arginine ester hydrolase activity with the synthetic substrate BAEE ([alpha]-N-benzoyl-L-arginine ethyl ester). The purified enzyme exhibited a molecular mass of 31.2 kDa on SDS-PAGE. When added to bovine plasma, the venom enzyme was able to liberate 0.70 moles of bradykinin per mole of enzyme in the isolated rat uterus assay. The venom enzyme, without an exogenous source of kininogen, did not cause an increase in capillary permeability as determined by the Evans blue stain method. The venom enzyme produced a drop of 7 mm Hg in systolic pressure when administered intravenously to a rabbit at 0.07 [micro]g/g body weight.

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Recent work has yielded kallikrein-like enzymes from the venoms of Crotalus adamanteus (cf. Markland et al. 1982), Crotalus atrox (cf. Bjarnason et al. 1983), Crotalus ruber ruber (cf. Mori & Sugihara 1988; 1989), Crotalus viridis viridis (cf. Komori et al. 1988), Agkistrodon piscivorus piscivorus (cf. Nikai et al. 1988) and Vipera aspis aspis (cf. Komori & Sugihara 1988). Kallikrein is known to possess arginine ester hydrolase activity and thus is able to cleave the ethyl ester group from the synthetic substrate BAEE ([alpha]-N-benzoyl-L-arginine ethyl ester). Many of these enzymes have also been functionally characterized for their kallikrein-like activity (i.e., their ability to release kinins, which cause contraction of visceral smooth muscle, an increase in capillary permeability and hypotension). Physiological assays have been developed to test each of these effects. This study presents findings on the isolation and functional characterization of a kallikrein-like enzyme from the venom of Crotalus basiliscus basiliscus.

Materials and Methods

Whole, lyophilized venom was obtained from Latoxan, lot number PA 346. Chromatography resins, [alpha]-N-benzoyl-L-arginine ethyl ester HCl (BAEE), bradykinin, kallikrein, bovine plasma and SDS-PAGE molecular weight standards were obtained from Sigma Chemical Company. SDS-PAGE pre-made gels were purchased from Bio-Rad. Solvents used for HPLC procedures were of HPLC grade. All other chemicals were of reagent grade quality.

BAEE activity. -- BAEE activity assays were performed by mixing 5-25 [micro]L of fractionated venom with 3 mL of buffered BAEE substrate (0.1 mg/mL BAEE buffered in a solution of 0.1 M Tris-HCI, pH 8.0 at ambient temperature). Activity was monitored with a Perkin-Elmer Lambda 7 UV/VIS spectrophotometer at 253 nm. BAEE hydrolase units are reported as the change in absorbance per minute per 25 [micro]L of enzyme solution.

Determination of molecular weight. -- SDS-polyacrylamide gel electrophoresis was carried out on 4-20% gradient and 12% non-gradient polyacrylamide Mini-PROTEAN II ready gels. Electrophoresis buffers, voltages, and coomassie brilliant blue staining and destaining were carried out according to instructions from the manufacturer.

Kinin-releasing activity. -- The assay method for kinin-releasing activity in the isolated rat uterus was adapted from Erspamer & Erspamer (1962), Trautschold (1970), and Komori et al. (1988). Venom enzyme (5.1 [micro]g/mL) mixed with an equal volume of bovine plasma was tested for the initiation of contractal events for smooth muscle preparatons. Uterine horn contractions were monitored on a myograph (Model F-60, Narco Bio-Systems) connected to a pen recorder (Narcotrace 40 physiograph, Narco Bio-Systems) set at a sensitivity of 50 mV/cm.

Capillary permeability-increasing activity. -- The assay method for capillary permeability-increasing activity was originally described by Miles & Wilhelm (1955) and later modified by Miller & Tu (1989), who used mice. Venom enzyme (2.0 and 2.5 [micro]g in 50 [micro]L), kallikrein (1.2, 2.5 and 25.0 [micro]g in 50 [micro]L) or whole venom (5.2 [micro]g in 50 [micro]L) was injected intradermally using a 1/2 inch-27 gauge needle.

Hypotensive activity. -- The procedure for this assay was adapted from Presley (1984:5-8) using the following Locke's solution (Lockwood 1961): NaCl, 9.0 g; KCl, 0.42 g; Ca[Cl.sub.2], 0.24 g; NaHC[O.sub.3], 0.15 g; glucose, 1.0 g/L; pH 7.3 at ambient temperature. Direct carotid blood pressure was measured in the urethane anesthetized New Zealand white rabbit. The carotid catheter was connected to a linear core pressure transducer (Model P-1000B, Narco Bio-Systems) set at 20 mV/cm sensitivity for direct measurement of blood pressure. Heart rate and respiration was monitored through thoracic needle electrodes coupled to a hi-gain coupler and an impedance pneumograph coupler, respectively.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

RESULTS

Isolation and molecular weight. -- 5.050 g of whole venom was dissolved in 45 mL of 50 mM Tris-HCl, 50 mM KCl buffer (pH 7.2 at 0-4[degrees] C), and applied to a Sephadex G-75 column (5.5 X 50 cm) equilibrated with the same buffer. A flow rate of 63 mL/hr at 0-4[degrees] C was maintained with the use of a peristaltic pump. Fractions were collected every 15 minutes. Fractions B1 (Fig. 1) were pooled, dialyzed, lyophilized and rechromatographed on a second G-75 column equilibrated with a buffer of greater ionic strength (50 mM Tris, 50 mM KCl, 100 mM N[H.sub.4]Ac, pH 7.2 at 0-4[degrees]C). The increase in ionic strength alleviated protein precipitation problems which were observed in the first G-75 column. Fractions containing 59% of the total BAEE hydrolase activity from the second G-75 column were pooled and rechromatographed on a third G-75 column (50 mM Tris-HCl, 50 mM KCl, pH 7.2 at 0-4[degrees]C).

Fractions containing 92% of the total BAEE hydrolase activity were applied to a DEAE Sephadex A-50 ion exchange column (2.6 X 81 cm) equilibrated with Tris buffer (50 mM Tris-HCl, 20 mM KCl, pH 7.2 at 0-4[degrees]C). The sample was eluted with an isocratic wash of approximately three bed volumes and then developed with a linear salt gradient to 1 M KCl.

Fraction E4 (Fig. 2) was then applied to a QAE Sephadex A-50 ion exchange column (2.6 X 79 cm) equilibrated and developed with buffer and salt gradient identical to the DEAE column.

Aliquots of fraction B5 (Fig. 3) were then applied to a an HPLC Aquapore CX-300 cation exchange column (220 X 4.6 mm) equilibrated with 5% buffer A (10 mM Tris-Ac, 0.2 M N[H.sub.4]Ac, pH 7.0 at ambient temperature) and 95% buffer B (10 mM Tris-Ac, pH 7.0 at ambient temperature). The column was developed with a linear gradient from 5% A to 60% A over 25-40 minutes with a flow rate of 1.0 mL/min and then re-equilibrated with 5% A and 95% B for ten minutes prior to the next run. Fractions B6 (Fig. 4), the kallikrein-like enzyme, were then collected and weighed.

SDS-PAGE of the purified enzyme revealed a single band corresponding to a molecular weight of 31.2 kDa.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Kinin-releasing activity in isolated rat uterus. -- Kinin-releasing activity assays showed that the venom enzyme (3.8 [micro]g, 130 picomoles, in 75 [micro]L), when added to an equal volume of plasma, induced contractions of the isolated rat uterus equal to 0.75 g force (Table 1). Comparison of the bradykinin dose-response curve with the venom enzyme-plasma mixture indicated that the enzyme produced a contraction force equal to the tissue response for 89 pmol of bradykinin. The enzyme, on a per mole basis, released kinin from the plasma corresponding to approximately 0.70 mol of bradykinin.

Capillary-permeability increasing activity. -- Intradermal injection of venom enzyme, without an exogenous source of kininogen, did not produce an increase in capillary permeability.

Hypotensive activity. -- Intravenous injections of venom enzyme resulted in a small decrease in mean arterial pressure followed by a rapid recovery (Figure 5).

DISCUSSION

The purified enzyme exhibited a molecular mass of 31.2 kDa. Recent studies have shown that other kallikrein-like enzymes from snake venoms have similar masses (Bjarnason et al. 1983; Komori et al. 1988; Mori & Sugihara 1988; Nikai et al. 1988).

The enzyme exhibited the ability to release kinin causing visceral smooth muscle contraction. The equivalent of 0.70 moles of bradykinin released per mole of enzyme indicates a moderate kinin-releasing ability.

[FIGURE 5 OMITTED]

An increase in capillary permeability was not seen following direct intradermal injection of either the venom enzyme or plasma kallikrein. The lack of any capillary permeability response may be due to the lack of sufficient endogenous kininogens in the injection area. Intradermal injections of whole venom did produce a noticible blue spot of 0.4 [cm.sup.2]. The whole venom activity is most likely due to the presence of spreading factors such as hemorrhagic enzymes.

In the preliminary studies, low concentrations of venom enzyme (0.07 [micro]g enzyme per gram of body weight) produced a 7 mm Hg decrease in systolic pressure. Comparisons of this low response to the results obtained with equal concentrations of bradykinin and kallikrein show that the enzyme does not produce a hypotensive kallikrein-like response in the rabbit model. Other kallikrein-like enzymes have been shown to cause sizable decreases in blood pressure resulting in prolonged hypotension in rats (Komori & Sugihara 1988; Komori et al. 1988; Mori & Sugihara 1988). These studies report the results from the administration of kallikrein-like enzymes at considerably higher concentrations than reported in this study. Further studies of the kallikrein-like enzyme concentration effects on blood pressure will be of considerable interest.
Table 1. Isolated rat uterus assay (kinin-releasing activity). Assay
substances at 110-130 pmol were mixed with an equivolume of bovine
plasma (150 [micro]L) unless noted otherwise and incubated at
37[degrees]C for five minutes.

Assay Mixture Uterus Contraction Bradykinine Moles of
 (g) quivalents bradykinin
 released (pmol) released/mole
 of substance

Enzyme + Plasma 0.75 89 0.70
Kallikrein* + Plasma 0.93 210 1.9
Enzyme only 0.00 0 0.00
Kallikrein only 0.00 0 0.00
Plasma only 0.00 0 0.00

* Porcine pancreatic kallikrein; molecular weight = 34 kDa (Fieldler,
1976)


ACKNOWLEDGEMENTS

This study was supported in part by a Robert A. Welch Foundation Grant to the Department of Chemistry. A portion of this study was used in partial fulfillment of the Master of Science degree, Midwestern State University, Wichita Falls, Texas.

LITERATURE CITED

Bjarnason, J. B., A. Barish, G. S. Direnzo, R. Campbell & J. W. Fox. 1983. Kallikrein-like enzymes from Crotalus atrox venom. J. Biol. Chem., 258: 12566-12573.

Erspamer, V., & G. F. Erspamer. 1962. Pharmacological actions of eledoisin on extravascular smooth muscle. Brit. J. Pharmacol., 19:337-354.

Fieldler, F. 1976. Pig pancreatic kallikreins a and b. Pp. 289-303, in Methods in enzymology (L. Lorand, ed.), Academic Press, New York, XLV Part B:XIX + 1-939 pp.

Komori, Y., & H. Sugihara. 1988. Physiological and biochemical properties of a kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper). Toxicon, 26:1193-1204.

______, T. Nikai & H. Sugihara. 1988. Biochemical and physiological studies on a kallikrein-like enzyme from the venom of Crotalus viridis viridis (Prairie rattlesnake). Biochimica et Biophysica Acta., 967:92-102.

Lockwood, A. P. M. 1961. "Ringer" solutions and some notes on the physiological basis of their ionic composition. Comp. Biochem. Physiol., 2:241-289.

Markland, F. S., C. Kettner, S. Schiffman, E. Shaw, S. S. Bajwa, K. N. N. Reddy, H. Kirakossian, G. B. Patkos, I. Theodor, and H. Pirkle. 1982. Kallikrein-like activity of crotalase, a snake venom enzyme that clots fibrinogen. Proc. Natl. Acad. Sci., USA, 79:1688-1692.

Miles, A. A., & D. L. Wilhelm. 1955. Enzyme-like globulins from serum reproducing the vascular phenomena of inflammation. I. An activable permeability factor and its inhibitor in guinea-pig serum. Br. J. Exp. Pathol., 36:71-81.

Miller, R. A., & A. T. Tu. 1989. Factors in snake venoms that increase capillary permeability. J. Pharm. Pharmcol., 41:792-794.

Mori, N., & H. Sugihara. 1988. Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom. Int. J. Biochem., 20:1425-1433.

______ & ______. 1989. Characterization of kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom. Int. J. Biochem., 21:83-90.

Nikai, T., K. Imai, M. Nagasaka & H. Sugihara. 1988. Kallikrein-like enzyme from the venom of Agkistrodon p. piscivorus. Int. J. Biochem., 20:1239-1245.

Presley, L. N. 1984. Some physiological effects of Crotalus basiliscus venom on the mammalian system. Unpubl. M. S. thesis, Midwestern State University, Wichita Falls, Texas, 48 pp.

Trautschold, I. 1970. Assay methods in the kinin system. Pp. 53-81, in Handbook of experimental pharmacology (E.G. Erdos, ed.), Springer-Verlag, Berlin, XXV:XIX+1-768 pp.

Robert D. Gaffin, Jon B. Scales and Rodney L. Cate

Department of Biology and Department of Chemistry, Midwestern State University, Wichita Falls, Texas 76308
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Author:Gaffin, Robert D.; Scales, Jon B.; Cate, Rodney L.
Publication:The Texas Journal of Science
Date:Feb 1, 1995
Words:2110
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