©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conversion of Bovine Pancreatic Phospholipase A at a Single Site into a Competitor of Neurotoxic Phospholipases A by Site-directed Mutagenesis (*)

(Received for publication, November 7, 1994)

Mu-Chin Tzeng (1) (2)(§) Chon-Ho Yen (1) Ming-Jhy Hseu (1) Cynthia M. Dupureur (3)(¶) Ming-Daw Tsai (3)(**)

From the  (1)Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 107, Republic of China, the (2)Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan, 107, Republic of China, and the (3)Department of Chemistry, The Ohio State University, Columbus, Ohio 43210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A(2) variants with neurotoxicity, including crotoxin, by chemical cross-linking experiments (Yen, C.-H., and Tzeng, M.-C.(1991) Biochemistry 30, 11473-11477). The binding of crotoxin to this receptor protein was completely suppressed by sufficient F22Y, a mutated bovine pancreatic phospholipase A(2) generated by site-directed mutagenesis of Phe of the wild-type enzyme to Tyr. The IC of this inhibition was estimated to be 1 µM. In sharp contrast, the wild-type enzyme gave no effect even at 50 µM. This mutation resulted in only minor and localized structural perturbations with little effect on enzymatic activity. Other phospholipase A(2) molecules capable of competing with crotoxin for this binding invariably have Tyr at this position. It was concluded that this Tyr residue is an important determinant for the binding of a number of phospholipase A(2) variants to the 45-kDa receptor.


INTRODUCTION

Proteins with phospholipase A(2) (PLA(2)) (^1)(EC 3.1.1.4) activity can be found in extracellular secretion as well as inside the cells of many organisms. The extracellular (secreted) PLA(2) variants exhibit a variety of biological effects, including phospholipid metabolism, host defense, signal transduction, neurotoxicity (presynaptic and/or postsynaptic), myotoxicity, and alteration of coagulation, which may or may not be related to hydrolysis of phospholipids. Despite large differences in biological actions, the secreted PLA(2) chains from most sources show high degrees of homology in the primary, secondary, and possibly tertiary structures. A small number of these proteins, including crotoxin from the South American rattlesnake Crotalus durissus terrificus, act primarily at the presynaptic level to cause synaptic blockade by inhibiting the release of neurotransmitters, though most of them also produce postsynaptic toxicity and other effects. To exert neurotoxicity, these PLA(2) neurotoxins appear to bind to the synaptic membranes strongly, followed by hydrolysis of the membrane phospholipids by the PLA(2) activity. Lack of such binding is apparently the reason why a larger number of PLA(2) variants, such as pancreatic PLA(2), are not neurotoxic despite high degrees of enzymatic activity. Strong binding to plasma membranes of other tissues may also be essential for the actions of many other PLA(2)s on these tissues (see (1, 2, 3, 4, 5, 6, 7, 8, 9) for recent reviews).

By the use of photoaffinity labeling and chemical cross-linking techniques, a few binding proteins have been identified for some of these presynaptic toxins(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . One subunit of the binding proteins for crotoxin and several other neurotoxic PLA(2)s is, as observed by us, a 45-kDa polypeptide preferentially present in the neuronal membrane(13) . As one of our approaches to understanding the structural basis for this binding, we converted bovine pancreatic PLA(2), which showed no detectable binding to the synaptic membrane, into a mutant capable of competing for the binding of crotoxin to this receptor by site-directed mutagenesis. This report is the first, to our knowledge, to convert pancreatic PLA(2) into a competitor of any neurotoxic PLA(2). This new way of using site-directed mutagenesis will be useful for the study of other toxic proteins as well.


EXPERIMENTAL PROCEDURES

Materials

Disuccinimidyl suberate and disuccinimidyl dithiobis(propionate) were obtained from Pierce. Na[I] and the kit for site-directed mutagenesis were supplied by Amersham Corp. Bovine pancreatic PLA(2) and the venom of C. durissus terrificus, from which crotoxin was purified as described (22) , were purchased from Sigma. Purification of rat PLA(2) from the pancreas was carried out according to published procedures (23) . All other chemicals were of reagent grade.

Generation and Purification of Mutant PLA(2)

Mutations of bovine pancreatic PLA(2) were generated by site-directed mutagenesis of a chemically synthesized gene for the wild-type protein (25) with an Amersham kit making use of the phosphorothioate method(24) . The procedures in the manufacturer's manual were followed. To construct F22Y and F22A mutants, oligonucleotides with the underscored bases in CAT AAT TGA AAA TAT CAA replaced by ATA and AGC, respectively, were used as primers. The mutated gene was subcloned into the expression vector pTO-N, and the resulting plasmid was transfected into a competent strain of Escherichia coli, BL21[pLysS]. The desired protein was purified after lysis of the bacteria by sonication (25, 26, 27) .

Cross-linking of I-Crotoxin to the Synaptic Membrane

Synaptic membrane fraction was purified from guinea pig brain by established methods(28, 29) . Crotoxin was labeled as previously described (13) with Na[I] using the chloramine-T method (30) to a specific activity of about 100 Ci/g protein (1 atom of iodine/molecule of crotoxin). I-Crotoxin was mixed with synaptic membranes (0.25 mg of protein/ml), with or without the presence of unlabeled pancreatic PLA(2), in 10 mM Tris-HCl buffer, pH 7.4, containing 0.5% bovine serum albumin, 150 mM NaCl, 10 mM SrCl(2), and 0.5 mM EGTA at 25 °C for 2.5 h. After dilution with 10 mM Tris-HCl, pH 7.4, the mixture was centrifuged at 8,000 times g for 10 min. The pellet was then washed and resuspended in phosphate-buffered saline. Disuccinimidyl suberate or disuccinimidyl dithiobis(propionate) in dimethyl sulfoxide was added to the resuspended membrane, and the mixture was incubated for 4 min at 25 °C. The reaction was stopped by adding 0.1 ml of 1 N glycine to each vial of the reaction mixture. After centrifugation at 15,000 times g for 15 min, the membrane pellet was solubilized with 0.1 M Tris-HCl buffer, pH 6.8, containing 5% glycerol and 2% SDS, and analyzed by SDS-polyacrylamide gel electrophoresis(31) . Precolored proteins (32) were used as molecular weight markers. After electrophoresis the position of the 60-kDa conjugate band was revealed by autoradiography. The corresponding area in the gel was cut off and counted by a -counter.

Toxicity Test

A test of the neurotoxicity of the PLA(2) mutants toward the central nervous system was performed by intracisternal injection of the proteins into mice weighing 22-25 g as described by Schanberg et al.(33) and Schweitz(34) . Peripheral toxicity was determined by intraperitoneal injection. Three dosage groups of mice with at least two animals in each group were given 1, 9, or 18 mg, respectively, of protein/kg of body weight in 20 µl of phosphate-buffered saline.

NMR Analysis

The enzyme sample (10 mg) was dissolved in 0.5 ml of D(2)O containing 300 mM NaCl and 50 mM CaCl(2) and then lyophilized. The residue was dissolved in 0.5 ml of 100% D(2)O, and the pH was adjusted to 4.0 with dilute DCl. Spectra at 500 MHz were recorded at 37 °C. Chemical shifts are relative to internal sodium 3-trimethylsilylpropionate-2,2,3,3-d(4). Other details are as described previously(35) .


RESULTS AND DISCUSSION

We labeled crotoxin with I without affecting its neurotoxicity. After I-crotoxin had been incubated with the synaptic membrane fraction from the brain to reach maximal binding, disuccinimidyl suberate or disuccinimidyl dithiobis(propionate) was employed to cross-link the binding complexes. Similar to results reported previously(13) , cross-linking with the above cross-linkers resulted in a 60-kDa conjugate when analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography (data not shown). Crotoxin is a complex of subunit A of 9,000 Da and subunit B of 14,400 Da in noncovalent association. The subunit B is an active PLA(2)(36, 37) .

Utilizing the phosphorothioate method(24) , we produced mutants of bovine pancreatic PLA(2) by site-directed mutagenesis of a chemically synthesized gene for the wild-type protein(25) . When the bovine pancreatic PLA(2) mutant F22Y, in which the Phe of the wild type is replaced by Tyr(35) , was present during the binding period, the subsequent formation of the 60-kDa radioactive conjugate was suppressed with an IC of 1 ± 0.1 µM as estimated from the curve in Fig. 1and three other separate experiments. In sharp contrast, the wild-type PLA(2) purified from the bovine pancreas gave no effect even at a concentration as high as 50 µM (Fig. 1). The wild-type pancreatic enzyme produced by cloning techniques and another mutant F22A, which has Ala at residue 22, were also without effect at the highest concentration used.


Figure 1: Effects of bovine pancreatic PLA(2) and the F22Y mutant on the cross-linking of I-crotoxin to synaptic membranes from the brain. Various amounts of the F22Y mutant (bullet) or the wild-type bovine pancreatic PLA(2) (circle) together with I-crotoxin (7 ng) were incubated with the synaptic membrane fraction (50 µg) from the guinea pig brain. After cross-linking, the counts of radioactivity in the 60-kDa conjugate were measured. The data are expressed as percentages of the counts in the absence of unlabeled PLA(2).



The one- and two-dimensional NMR spectra of F22Y and the wild type are almost identical except for the obvious changes arising from the new phenolic OH group and a 0.19 ppm change in one of the three chemical shifts of Phe, which is in close proximity to residue 22, forming the second half of the Phe-Phe aromatic sandwich ( Fig. 2and Fig. 3and Table 1). Similarly, except right around the mutated residue, the NMR spectra of F22A are perturbed only slightly(35) . The enzymatic activity of the two mutants is also comparable with that of the wild-type enzyme(35) . Among the many mutants we have analyzed, F22Y is most similar to the wild type structurally and kinetically. Hence it is unlikely that the inhibitory effect of the F22Y mutant is due to the hydrolysis of membrane phospholipids. In addition, we have chosen the assay condition that minimizes the enzymatic activity by using a solution containing 10 mM Sr, 0.5 mM EGTA, and no Ca, as it has been shown that Sr is antagonistic to Ca (see (1, 2, 3, 4) for reviews), which is required for the enzymatic activity of secreted PLA(2). Moreover, F22Y was equally effective in suppressing the formation of the 60-kDa conjugate when the experiments were performed at 4 °C to completely arrest the enzymatic activity. We thus conclude that the F22Y mutant blocked the formation of the radioactive conjugate by competing the binding of I-crotoxin to the binding protein.


Figure 2: One-dimensional proton NMR spectra of pancreatic PLA(2) and its F22Y mutant in D(2)O. Spectra of samples of 1.5 mM protein in 300 mM NaCl and 50 mM CaCl(2), pH 4.0, were recorded at 500 MHz and 37 °C. Free induction decays from 200 scans were processed with gaussian multiplications (line broadening, -5; gaussian broadening, 0.1). WT, wild type.




Figure 3: Phase-sensitive nuclear Overhauser effect spectroscopy proton NMR spectra in D(2)O at 500 MHz. Samples are as described in the legend of Fig. 2. The mixing time was 200 ms. A 4096 times 512 matrix in the time domain was recorded and zero-filled to a 4096 times 2048 matrix prior to multiplication by a gaussian function (line broadening, -3; Gaussian broadening, 0.1). Chemical shifts for the indicated spin systems are given and assigned in Table 1.





However, because the binding affinity of the F22Y mutant was not high relative to that of crotoxin (<10 nM), there must be other residues also involved in binding and thereby in neurotoxicity. Judging from the affinity of F22Y, we would not expect it to be neurotoxic. All mice injected with the F22Y mutant, either intraperitoneally or intracisternally, lived and behaved normally even at a dose of 18 mg of protein/kg of body weight. We have iodinated the F22Y mutant and attempted to demonstrate its binding to the synaptic membrane directly. Specific binding was not evident, apparently because the affinity is too low.

We were also aware that residue 22 of the pancreatic PLA(2) of the rat is Tyr. If the rat enzyme blocked I-crotoxin from forming the radioactive conjugate, our conclusion would be further substantiated. We therefore purified the PLA(2) from the rat pancreas and then investigated its effect on the conjugation. As expected, the rat pancreatic PLA(2) could completely inhibit the generation of the 60-kDa band, although the potency (IC = 10 µM) was lower than that of the F22Y mutant of the bovine PLA(2). This may be due to the differences between the rat and the bovine enzymes at other areas. As would be expected, we found that the rat PLA(2) was nontoxic when tested as described above.

Although further work is needed to extend our findings for a generalization concerning the binding of the neurotoxic PLA(2)s, it would be useful to put forward some suggestions for further testing. It has not been possible for us to study the binding of all variants of PLA(2), but available data seem to indicate that the 45-kDa polypeptide may be a common binding protein, or one of its subunits, for most neurotoxic PLA(2)s, and strong binding to this polypeptide appears to be linked to neurotoxicity (see (1) for review; (11) and (13) ). Significantly, examination of the aligned sequences of the PLA(2) chains from various sources (38, 39) revealed that the active PLA(2) chain of each of the PLA(2) neurotoxins has Tyr at the position corresponding to Phe of the bovine enzyme in their aligned sequences (it is position 21 for many PLA(2) chains, including subunit B of crotoxin), whereas those with Phe are nontoxic or marginally toxic. Our present results would suggest that Tyr in these neurotoxic PLA(2) variants is also an important determinant for such binding and that other as yet undefined determinants are also involved. On such ground, one may resolve the seeming conflict that, though a small number of PLA(2)s with Tyr exhibit low toxicity, most of them are potent toxins. With Tyr in its PLA(2) chain, beta-bungarotoxin is to date the only neurotoxic PLA(2) that does not bind to the 45-kDa polypeptide. This can be explained by steric hindrance due to another polypeptide covalently bonded to its PLA(2) chain, an interpretation supported by the insusceptibility of this residue to chemical modification(40) . As to the other determinants for binding, a definitive answer has not been obtained for any of the PLA(2) variants, but some information is available (see (41) for review). Based on comparisons of the amino acid sequences of the PLA(2) variants with presynaptic toxicity and those of the nontoxic ones, basic residues around position 59, at position 69, around position 76, and at position 93 (or 94) and the segments of residues 1-7, 68-85, and 80-110 (all numbered with respect to pancreatic PLA(2) according to (42) ) have been proposed to be involved in binding(43, 44) . From the variations in amino acid sequences of the three ammodytoxins, Tyr, Arg, and Lys have been suggested to participate in binding(45, 46) . Chemical modification and other studies indicate that Trp, Tyr^7, Tyr and Tyr of notexin, Tyr of the PLA(2) chain of beta-bungarotoxin, and Asn^1, Met^8, Lys, and Lys of Pa-11 are probably needed for their neurotoxicity, but binding experiments have not been performed except for some modified Pa-11(40, 47, 48, 49, 50) . Extension of the site-directed mutagenesis studies that we have reported here will hopefully provide a clear-cut answer.

In summary, we have identified Tyr as one of the important determinants for the neurotoxicity of PLA(2) proteins. The Phe of bovine pancreatic PLA(2) is located at the B-helix, away from the active site and exposed to solvent. It does not play an important structural role because mutations at this position cause only minor and localized structural perturbations. The F22Y is a silent mutation catalytically, but it acquires the ability to compete with crotoxin (a neurotoxic PLA(2)) for binding to its receptor protein with an IC value of 1 µM. Although such a binding affinity is still too low for in vivo neurotoxicity (the affinity of crotoxin for binding to the same receptor protein is <10 nM), the results have demonstrated a dramatic effect that can possibly be induced by a point mutation and have shed light on how the small PLA(2) protein (14 kDa for pancreatic enzymes) could give rise to hundreds of natural variants displaying a great variety of pharmacological actions.


FOOTNOTES

*
This work was supported in part by Grant NSC82-0211-B001-041 from the National Science Council, R.O.C. (to M.-C. T.). This is the thirteenth paper in the series ``Phospholipase A(2) Engineering'' supported by Grant GM41788 from the National Institutes of Health (to M.-D. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests may be addressed: Inst. of Biological Chemistry, Academia Sinica, P. O. Box 23-106, Taipei, Taiwan, 107, R.O.C. Fax: 886-23635038.

Current address: Dept. of Chemistry, California Inst. of Technology, Pasadena, CA 91125.

**
To whom reprint requests may be addressed: Dept. of Chemistry, Ohio State University, 120 W. 18th Ave., Columbus, OH 43210.

(^1)
The abbreviation used is: PLA(2), phospholipase A(2).


ACKNOWLEDGEMENTS

We thank Hongxin Zhu for assistance in purifying F22Y and S. H. Rao for typing the manuscript.


REFERENCES

  1. Tzeng, M.-C. (1993) J. Toxicol. Toxin Rev. 12, 1-62
  2. Harris, J. B. (1991) in Snake Toxins (Harvey, A. L., ed) pp. 91-129, Pergamon Press, Inc., Tarrytown, NY
  3. Hawgood, B., and Bon, C. (1991) in Handbook of Natural Toxins, Vol. 5: Reptile Venoms and Toxins (Tu, A. T., ed) pp. 3-52, Marcel Dekker, Inc., New York
  4. Davidson, F. F., and Dennis, E. A. (1991) in Handbook of Natural Toxins, Vol. 5: Reptile Venoms and Toxins (Tu, A. T., ed) pp. 107-145, Marcel Dekker, Inc. New York
  5. Harvey, A. L. (1990) Int. Rev. Neurobiol. 32, 201-239 [Medline] [Order article via Infotrieve]
  6. Rosenberg, P. (1990) in Handbook of Toxinology (Shier, W. T., and Mebs, D., eds) pp. 67-277, Marcel Dekker, Inc., New York
  7. Dennis, E. A. (1994) J. Biol. Chem. 269, 13057-13060 [Free Full Text]
  8. Mayer, R. J., and Marshall, L. A. (1993) FASEB J. 7, 339-348 [Abstract/Free Full Text]
  9. Kudo, I., Murakami, M., Hara, S., and Inoue, K. (1993) Biochim. Biophys. Acta 1170, 217-231 [Medline] [Order article via Infotrieve]
  10. Tzeng, M.-C., Hseu, M. J., Yang, J. H., and Guillory, R. J. (1986) J. Protein Chem. 5, 221-228
  11. Tzeng, M.-C., Hseu, M. J., and Yen, C.-H. (1989) Biochem. Biophys. Res. Commun. 165, 689-694 [Medline] [Order article via Infotrieve]
  12. Hseu, M. J., Guillory, R. J., and Tzeng, M.-C. (1990) J. Bioenerg. Biomembr. 22, 39-50 [Medline] [Order article via Infotrieve]
  13. Yen, C.-H., and Tzeng, M.-C. (1991) Biochemistry 30, 11473-11477 [Medline] [Order article via Infotrieve]
  14. Othman, I. B., Spokes, J. W., and Dolly, J. O. (1982) Eur. J. Biochem. 128, 267-276 [Abstract]
  15. Rehm, H., and Betz, H. (1982) J. Biol. Chem. 257, 10015-10022 [Abstract/Free Full Text]
  16. Rehm, H., and Betz, H. (1983) EMBO J. 2, 1119-1122 [Medline] [Order article via Infotrieve]
  17. Rehm, H., and Lazdunski, M. (1988) Biochem. Biophys. Res. Commun. 153, 231-240 [Medline] [Order article via Infotrieve]
  18. Lambeau, G., Barhanin, J., Schweitz, H., Qar, J., and Lazdunski, M. (1989) J. Biol. Chem. 264, 11503-11510 [Abstract/Free Full Text]
  19. Lambeau, G., Schimd-Alliana, A., Lazdunski, M., and Barhanin, J. (1990) J. Biol. Chem. 265, 9526-9532 [Abstract/Free Full Text]
  20. Scott, V. E. S., Parcej, D. N., Keen, J. N., Findlay, J. B. C., and Dolly, J. O. (1990) J. Biol. Chem. 265, 20094-20097 [Abstract/Free Full Text]
  21. Degn, L. L., Seebart, C. S., and Kaiser, I. I. (1991) Toxicon 29, 973-988 [Medline] [Order article via Infotrieve]
  22. Hendon, R. A., and Tu, A. T. (1979) Biochim. Biophys. Acta 578, 243-252 [Medline] [Order article via Infotrieve]
  23. Ono, T., Tojo, H., Inoue, K., Kagamijama, H., Yamano, T., and Okamoto, M. (1984) J. Biochem. (Tokyo) 96, 785-792 [Abstract]
  24. Sayers, J. R., Krekel, C., and Eckstein, F. (1992) BioTechniques 13, 592-596 [Medline] [Order article via Infotrieve]
  25. Noel, J. P., and Tsai, M.-D. (1989) J. Cell. Biochem. 40, 309-320 [Medline] [Order article via Infotrieve]
  26. Deng, T., Noel, J. P., and Tsai, M.-D. (1990) Gene (Amst.) 93, 229-234 [CrossRef][Medline] [Order article via Infotrieve]
  27. Noel, J. P., Bingman, C. A., Deng, T., Dupureur, C. M., Hamilton, K. J., Jiang, R. T., Kwak, J.-G., Sekharudu, C., Sundaralingam, M., and Tsai, M.-D. (1991) Biochemistry 30, 11801-11811 [Medline] [Order article via Infotrieve]
  28. Whittaker, V. P. (1959) Biochem. J. 72, 694-706 [Medline] [Order article via Infotrieve]
  29. De Robertis, E., Rodriguez de Lores Arnaiz, G., Salganicoff, L., Pellegrino de Iraldi, A., and Zieher, L. M. (1963) J. Neurochem. 10, 225-235 [Medline] [Order article via Infotrieve]
  30. Hunter, W. M., and Greenwood, F. C. (1962) Nature 194, 495-496
  31. Neville, D. M., Jr. (1971) J. Biol. Chem. 246, 6328-6334 [Abstract/Free Full Text]
  32. Tzeng, M.-C. (1983) Anal. Biochem. 128, 412-414 [Medline] [Order article via Infotrieve]
  33. Schanberg, S. M., Schildkraut, J. J., and Kopin, I. J. (1967) J. Pharmacol. Exp. Ther. 157, 311-318 [Medline] [Order article via Infotrieve]
  34. Schweitz, H. (1984) Toxicon 22, 308-311 [CrossRef][Medline] [Order article via Infotrieve]
  35. Dupureur, C. M., Yu, B. Z., Mamone, J. A., Jain, M. K., and Tsai, M.-D. (1992) Biochemistry 31, 10576-10583 [Medline] [Order article via Infotrieve]
  36. Hendon, R. A., and Fraenkel-Conrat, H. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 1560-1563 [Abstract]
  37. Rübsamen, K., Breithaupt, H., and Habermann, E. (1971) Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. 270, 274-288
  38. Mebs, D., and Klaus, I. (1991) in Snake Toxins (Harvey, A. L., ed) pp. 425-447, Pergamon Press, Inc., Tarrytown, NY
  39. Heinrikson, R. L. (1991) Methods Enzymol. 197, 201-214 [Medline] [Order article via Infotrieve]
  40. Yang, C. C., and Lee, H. J. (1986) J. Protein Chem. 5, 15-28
  41. Yang, C. C. (1994) J. Toxicol. Toxin Rev. 13, 125-177
  42. Renetseder, R., Brunie, S., Dijkstra, B. W., Drenth, J., and Sigler, P. B. (1985) J. Biol. Chem. 260, 11627-11634 [Abstract/Free Full Text]
  43. Kini, R. M., and Iwanaga, S. (1986) Toxicon 24, 527-541 [Medline] [Order article via Infotrieve]
  44. Tsai, I. H., Liu, H. C., and Chang, T. (1987) Biochim. Biophys. Acta 916, 94-99 [Medline] [Order article via Infotrieve]
  45. Ritonja, A., Machleidt, W., Turk, V., and Gubensek, F. (1986) Biol. Chem. Hoppe-Seyler 367, 919-923 [Medline] [Order article via Infotrieve]
  46. Krizaj, I., Turk, D., Ritonja, A., and Gubensek, F. (1989) Biochim. Biophys. Acta 999, 198-202 [Medline] [Order article via Infotrieve]
  47. Mollier, P., Chwetzoff, S., Bouet, F., Harvey, A. L., and Menez, A. (1989) Eur. J. Biochem. 185, 263-270 [Abstract]
  48. Yang, C. C., and Chang, L.-S. (1991) Biochem. J. 280, 739-744 [Medline] [Order article via Infotrieve]
  49. Tsai, I. H., and Tzeng, M.-C. (1991) in Peptides: Chemistry and Biology (Smith, J. A., and Rivier, J. E., eds) pp. 460-461, ESCOM Science Publishers, Leiden, The Netherlands
  50. Takasaki, C., Sugama, A., Yanagita, A., Tamiya, N., Rowan, E. G., and Harvey, A. L. (1990) Toxicon 28, 107-117 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.




This Article
Abstract
Full Text (PDF)
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Copyright Permissions
Google Scholar
Articles by Tzeng, M.-C.
Articles by Tsai, M.-D.
Articles citing this Article
PubMed
PubMed Citation
Articles by Tzeng, M.-C.
Articles by Tsai, M.-D.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   Biochemistry and Molecular Biology Education 
Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.