Advertisement

Journal of Comparative Physiology B

, Volume 155, Issue 2, pp 135–143 | Cite as

Isolation and characterization of plasma coagulogen (PC) of the cockroachLeucophaea maderae (Blattaria)

  • Barbara Barwig
Article

Summary

  1. 1.

    The plasma coagulogen (PC), one of the two clotting proteins of the African cockroachLeucophaea maderae, was obtained from the hemolymph of last nymphal instars, isolated and further analyzed.

     
  2. 2.

    The isolation of plasma coagulogen was carried out by two methods, (i) dissociation of plasma coagulogen from an association of plasma coagulogen and insoluble hemocyte coagulogen, and (ii) precipitation with buffer of low ionic strength.

     
  3. 3.

    The purified protein was homogeneous when tested by polyacrylamide gel electrophoresis, immunoelectrophoresis and sedimentation analysis.

     
  4. 4.

    PC is a lipoprotein with a lipid content of 50%. A carbohydrate component of about 1% was also detected. The molecular weight of the lipoprotein is about 640,000. The sedimentation coefficient was determined to be 7.7 S.

     
  5. 5.

    SDS-PAGE of plasma coagulogen revealed two subunits: a heavy chain (PC1) with a molecular weight of about 320,000 and a light chain (PC2), with 76,000.

     
  6. 6.

    The amino acid analysis indicated high glutamine/glutamic acid as well as asparagine/aspartic acid values.

     
  7. 7.

    The lipid component showed a high percentage of phospholipids and diglycerides.

     
  8. 8.

    PC is suggested to be identical with lipophorin because of its high similarity with other insect lipophorins.

     

Keywords

Lipid Carbohydrate Polyacrylamide Ionic Strength Lipid Content 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

DTT

dl-dithiothreitol

EDTA

ethylenediamine-tetraacetic acid, disodium salt

HC

hemocyte coagulogen

PAGE

polyacrylamide gel electrophoresis

PAS

periodic acid-Schiff reagent

PC

plasma coagulogen

SDS

sodium dodecyl sulfate

TLC

thin layer chromatography

TLG

thin layer gel filtration

\(\bar v\)

partial specific volume

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Andrews P (1970) Estimation of molecular size and molecular weights of biological compounds by gel filtration. Meth Biochem Anal 18:1–53Google Scholar
  2. Barwig B, Bohn H (1980) Evidence for the presence of two clotting proteins in insects. Naturwissenschaften 67:47Google Scholar
  3. Bohn H (1977) Differential adhesion of the hemocytes ofLeucophaea maderae (Blattaria) to a glass surface. J Insect Physiol 23:185–194Google Scholar
  4. Bohn H, Barwig B (1984) Hemolymph clotting in the cockroachLeucophaea maderae (Blattaria). Influence of ions and inhibitors; isolation of the plasma coagulogen. J Comp Physiol B 154:457–467Google Scholar
  5. Bohn H, Barwig B, Bohn B (1981) Immunochemical analysis of hemolymph clotting in the insectLeucophaea maderae (Blattaria). J Comp Physiol 143:169–184Google Scholar
  6. Bowen TJ, Kilby BA (1953) Electrophoresis of locust hemolymph. Arch Int Physiol 61:413–416Google Scholar
  7. Brehélin M (1972) Étude du mécanisme de la coagulation de l'hémolymphe d'un Acridien:Locusta migratoria migratorioides (R.&F.). Acrida 1:167–175Google Scholar
  8. Brehélin M (1979) Hemolymph coagulation inLocusta migratoria: Evidence for a functional equivalent of fibrinogen. Comp Biochem Physiol 62B:329–334Google Scholar
  9. Chino H, Katalase H, Downer RGH, Takahashi K (1981) Diacylglycerol-carrying lipoprotein of hemolymph of the American cockroach: purification and function. J Lipid Res 22:7–15Google Scholar
  10. Chino H, Kitizawa K (1981) Diacylglycerol-carrying lipoprotein of hemolymph of the locust and some insects. J Lipid Res 22:1042–1052Google Scholar
  11. Chino HS, Murakami S, Harashima K (1969) Diglyceride-carrying lipoproteins in insect hemolymph: isolation, purification and properties. Biochim Biophys Acta 176:1–26Google Scholar
  12. Chino H, Yamagata M, Takahashi K (1976) Isolation and characterization of insect vitellogenin. Its identity with hemolymph lipoprotein II. Biochim Biophys Acta 441:349–353Google Scholar
  13. Clausen J (1971) Immunochemical techniques for the identification and estimation of macromolecules. In: Work TS, Work E (eds) Laboratory techniques in biochemistry and molecular biology. North-Holland, Amsterdam, pp 405–556Google Scholar
  14. Cohn EJ, Edsall JT (1950) Proteins, amino acds and peptides. Reinhold, New York, pp 375Google Scholar
  15. Engelmann F, Friedel T, Ladduwahetty M (1976) The native vitellogenin of the cockroachLeucophaea maderae. Insect Biochem 6:211–220Google Scholar
  16. Folch I, Lees M, Sloane-Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509Google Scholar
  17. Freemann CP, West D (1966) Complete separation of lipid classes on a single thin-layer plate. J Lipid Res 7:324–327Google Scholar
  18. Gellissen G (1983) Lipophorin as the plasma coagulogen inLocusta migratoria. Naturwissenschaften 70:45–46Google Scholar
  19. Gellissen G, Emmerich H (1980) Purification and properties of a digylceride-binding lipoprotein (LPI) of the hemolymph of adult maleLocusta migratoria. J Comp Physiol 136:1–9Google Scholar
  20. Gellissen G, Wyatt GR (1981) Production of lipophorin in the fat body of adultLocusta migratoria: comparison with vitellogenin. Can J Biochim 59:648–654Google Scholar
  21. Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  22. Landureau J-C, Grellet F (1972) Nouvelles techniques de culture in vitro de cellules d'insectes et leures applications. CR Acad Sci (Paris) 274:1372–1375Google Scholar
  23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  24. Pattnaik NM, Mundall EC, Trambusti BG, Law JH, Kézdy FJ (1979) Isolation and characterization of a larval lipoprotein from the hemolymph ofManduca sexta. Comp Biochem Physiol 63B:469–476Google Scholar
  25. Peled Y, Tietz A (1975) Isolation and properties of a lipoprotein from the hemolymph of the locust,Locusta migratoria. Insect Biochem 5:413–416Google Scholar
  26. Prat JP, Lamy JN, Weill JD (1969) Coloration des lipoproteines après électrophorèse en gel de polyacrylamide. Bull Soc Chim Biol 51:1367Google Scholar
  27. Roe JR (1955) The determination of sugar in blood and spinal fluid with the anthrone reagent. J Biol Chem 212:335–343Google Scholar
  28. Siakotos AN (1960) The conjugated plasma proteins of the American cockroach. II. Changes during the molting and clotting process. J Gen Physiol 43:1015–1030Google Scholar
  29. Thomas KK, Gilbert LI (1968) Isolation and characterization of the hemolymph lipoproteins of the American silkmoth,Hyalophora cepropia. Arch Biochem Biophys 127:512–521Google Scholar
  30. Weber K, Osborn M (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412Google Scholar
  31. Yphantis DA (1960) Rapid determination of molecular weights of peptides and proteins. Ann NY Acad Sci 88:586–601Google Scholar
  32. Yphantis DA (1964) Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297–317Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Barbara Barwig
    • 1
  1. 1.Zoologisches Institut der UniversitätMünchen 2Federal Republic of Germany

Personalised recommendations