Skip to main content
SpringerLink
Log in

Interaction of creatine kinase and hexokinase with the mitochondrial membranes, and self-association of creatine kinase: crosslinking studies

  • Original Article
  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Summary

Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.

The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.

Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.

Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu++-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.

In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AK:

Adenylate Kinase (EC 2.7.4.3)

CKm:

Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2)

DMS:

Dimethyl Suberimidate

DTT:

Dithiothreitol

EGS:

Ethylene Glycol bis (succinimidyl succinate)

EGTA:

Ethylene Glycol bis (aminoethyl ether)

N,N,N′,N′:

Tetraacetic acid

G6P:

Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N′-2 Ethane Sulfonic Acid

HK:

Hexokinase (EC 2.7.1.1)

MABI:

methyl 4-Azido Benzoimidate

NaPi:

Sodium Phosphate

SANPAH:

N-Succinimidyl 6(4′ azido 2′ nitrophenylamino) Hexanoate

SDS:

Sodium Dodecyl Sulfate (sodium lauryl sulfate)

Tris:

Tris (hydroxymethyl) Aminomethane

References

  1. Bessman SP, Geiger PJ: Compartmentation of hexokinase and creatine phosphokinase, cellular regulation and insulin action. In: Current Topics in Cellular Regulation Vol 16, pp 55–86, 1980

    Google Scholar 

  2. Felgner PL, Meser JL, Wilson DE: Purification of a hexokinase binding protein from the outer mitochondrial membrane. J Biol Chem 254:4946–4949, 1979

    Google Scholar 

  3. Fieck C, Benz R, Roos N, Brdiczka D: Evidence for identity between the hexokinase binding protein and the mitochondrial porin in the outer membrane of rat liver mitochondria. Biochim Biophys Acta 688:429–440, 1982

    Google Scholar 

  4. Linden M, Gellefors P, Nelson BD: Pore protein and the hexokinase binding protein from the outer membrane of rat liver mitochondria are identical. FEBS Letters 141:189–192, 1982

    Google Scholar 

  5. Gellerich F, Saks VA: Control of heart mitochondrial oxygen consumption by creatine kinase: the importance of enzyme localization. Biochem Biophys Res Commun 105:1473–1482, 1982

    Google Scholar 

  6. Muller M, Moser R, Cheneval D, Carafoli E: Cardiolipin is the membrane receptor for mitochondrial creatine kinase, J Biol Chem 260:3839–3843, 1985

    Google Scholar 

  7. Ji TH: The application of chemical crosslinking for studies on cell membranes and the identification of surface reporters. Biochim Biophys Acta 559:39–79, 1979

    Google Scholar 

  8. Gaffney BJ: Chemical and biochemical crosslinking of membrane components. Biochim Biophys Acta 822:289–317, 1985

    Google Scholar 

  9. Font B, Vial C, Goldschmidt D, Eichenberger D, Gautheron DC: Heart mitochondrial creatine kinase solubilization. Effect of mitochondrial swelling and SH group reagents. Arch Biochem Biophys 212:195–203, 1981

    Google Scholar 

  10. Deluca M, Hall N: Studies of the properties of purified mitochondrial creatine kinase. In: WE Jacobus, JS Ingwall (eds). Heart creatine kinase. Williams and Wilkins Baltimore (1980), pp 18–27

    Google Scholar 

  11. Aubert-Foucher E, Font B, Gautheron DC: Modified properties of hexokinase from heart mitochondria prepared using proteolytic enzyme. Mol Cell Biochem 67:111–118, 1985

    Google Scholar 

  12. Vial C, Marcillat O, Goldschmidt D, Font B, Eichenberger D: Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts. Arch Biochem Biophys 251:558–566, 1986

    Google Scholar 

  13. Aubert-Foucher E, Font B, Gautheron DC: Rabbit heart mitochondrial hexokinase: solubilization and general properties. Arch Biochem Biophys 232:391–399, 1984

    Google Scholar 

  14. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680–685, 1970

    PubMed  Google Scholar 

  15. Weber K, Osborn H: The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis, J Biol Chem 244:4406–4412, 1969

    Google Scholar 

  16. Peters K, Richards FM: Chemical crosslinking reagents and problems in studies of membrane structure. Ann Rev Biochem 46:523–551, 1977

    Google Scholar 

  17. Font B, Gautheron DC: General and kinetic properties of pig heart mitochondrial adenylate kinase. Biochim Biophys Acta 611:299–308, 1980

    Google Scholar 

  18. Davies GE, Stark GR: Use of dimethyl suberimidate a crosslinking reagent in studying the subunit structure of oligomeric proteins. Proc Nat Acad Sci USA, 66:651–656, 1970

    Google Scholar 

  19. Carpenter FH, Harrington KT: Intermolecular crosslinking of monomeric proteins and crosslinking of oligomeric proteins as a probe of quaternary structure. J Biol Chem 247:5580–5586, 1972

    Google Scholar 

  20. Hucho F, Muller H, Sund H: Investigation of the symmetry of oligomeric enzymes with bifunctional reagents. Eur J Biochem 59:79–87, 1975

    Google Scholar 

  21. Hajdu J, Bartha F, Friedrich P: Crosslinking with bifunctional reagents as a mean for studying the symetry of oligomeric proteins. Eur J Biochem 68:373–383, 1976

    Google Scholar 

  22. Lambin P: Reliability of molecular weight determination of proteins by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Anal Biochem, 85:114–125, 1978

    Google Scholar 

  23. Jacobs HK, Graham M: Physical and chemical characterization of mitochondrial creatine kinase from bovine heart. Fed Proc 37:1574, 1978

    Google Scholar 

  24. Lipskaya TY, Kedishvili NY, Kalenova ME: Conditions of interconversion of oligomeric forms of heart mitochondrial creatine kinase. Biochemistry USSR, 50:1339–1348, 1985

    Google Scholar 

  25. Walliman Th, Zurbriggen B, Walzthongy D, Eppenberger HM: Native mitochondrial creatine kinase from chicken heart is an octamer. J Muscle Res and Cell Motility. 7 Abstract No 40B, 1986

  26. Middaugh CR, Vanin EF, Ji Th: Chemical crosslinking of cell membrances. Mol Cell Biochem 50:115–141, 1983

    Google Scholar 

  27. Krause J, Hay R, Kowollik C, Brdiczka D: Cross-linking analysis of yeast mitochondrial outer membrane. Biochim Biophys Acta, 860:690–698, 1986

    Google Scholar 

  28. Rendon A, Rott R, Avi-Dor Y: Proton permeation adenine nucleotide translocation and respiratory control in mitochondria crosslinked by dimethylsuberimidate. Biochim Biophys Acta 570:290–299, 1980

    Google Scholar 

  29. D'souza SF, Srere PA: Crosslinking of mitochondrial matrix proteins in situ. Biochim Biophys Acta 724:40–51, 1983

    Google Scholar 

  30. Salacinski PRP, McLean C, Shyes JEC, Clement-Jones VV, Lowy PJ: Iodination of proteins, glycoproteins and peptides using a solid-phase oxidizing agent, 1, 3, 4, 6 tetrachloro-3, 6 diphenyl glycoluril (Iodogen). Anal Biochem 117:136–146, 1981

    Google Scholar 

  31. Tarvers RC, Roberts HR, Lundblad BL: Self association of bovine prothrombin fragment 1 in the presence of metal ions. Use of a covalent crosslinking reagent to study the reaction. J Biol Chem 259:1944–1950, 1984

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratoire de Biologie et de Technologie des Membranes du C.N.R.S, Villeurbanne, France

    Bernard Font, Denise Eichenberger, Denise Goldschmidt & Christian Vial

Authors
  1. Bernard Font
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Denise Eichenberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Denise Goldschmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christian Vial
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Font, B., Eichenberger, D., Goldschmidt, D. et al. Interaction of creatine kinase and hexokinase with the mitochondrial membranes, and self-association of creatine kinase: crosslinking studies. Mol Cell Biochem 78, 131–140 (1987). https://doi.org/10.1007/BF00229687

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00229687

Key words

Search

Navigation