Isolation and properties of creatine kinase from the breast muscle of tropical fruit bat, Eidolon helvum (kerr)

Afolayan, Adeyinka; Daini, OA

doi:10.1016/0305-0491(86)90028-3

Cited by 2 publications

(1 citation statement)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To address the question of whether Cr uptake into mitochondria could be due to simple diffusion through the inner mitochondrial membrane, or whether it is indeed mediated by a transporter protein, we tested the effect of different sulfhydryl-modifying reagents, like DTNB, DNFB, and NEM. These reagents have been tested earlier for their ability to inhibit enzymatic activities at rather low concentrations (25)(26)(27). In our experiments, DTNB and DNFB had rather little effect on Cr uptake.…”

Section: Fig 3 Immunoreactivity Of Rabbit Anti-crt Antibodiesmentioning

confidence: 52%

Novel Mitochondrial Creatine Transport Activity

Walzel¹,

Speer²,

Zanolla³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N-and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of ϳ70 and ϳ55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [ 14 C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentrationdependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

show abstract

Section: Fig 3 Immunoreactivity Of Rabbit Anti-crt Antibodiesmentioning

confidence: 52%

Novel Mitochondrial Creatine Transport Activity

Walzel¹,

Speer²,

Zanolla³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Creatine transporters: A reappraisal

et al. 2004

View full text Add to dashboard Cite

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na(+)/Cl(-)-dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain. In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (alpha-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the alpha-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our alpha-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of approximately 60 kDa was enriched. This latter is most likely representing the genuine plasma membrane CrT. Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehyd...

show abstract

Isolation and properties of creatine kinase from the breast muscle of tropical fruit bat, Eidolon helvum (kerr)

Cited by 2 publications

References 14 publications

Novel Mitochondrial Creatine Transport Activity

Novel Mitochondrial Creatine Transport Activity

Creatine transporters: A reappraisal

Contact Info

Product

Resources

About