Presence of thiamine pyrophosphate in mammalian peroxisomes

Abstract

Background: Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the alpha-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.

Results: Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 +/- 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.

Conclusion: Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.

Figure 2

Subcellular distribution of TPP and TPK activity. The nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and cytosolic (S) fractions, obtained by fractionation of a fresh rat liver homogenate, were analyzed for TPP content (panel A), TPK activity (panel B) and lactate dehydrogenase, marker enzyme for cytosol (panel C, LDH) and other marker enzymes (data not shown). Results are expressed as relative specific activities versus percentage of total protein. Relative specific activity is defined as the percentage of total recovered activity present in a particular fraction divided by the corresponding percentage of protein. Recoveries for TPP content (23 nmol/g liver) and LDH (456 U/g liver) and TPK (263 nmol/g liver/h) activities were 128%, 97% and 97%, respectively. Recoveries for GDH activity and protein content were 102% and 94%, respectively (data not shown). A second fractionation (data not shown) resulted in a similar histogram for TPP (28 nmol/g liver), but with somewhat more enrichment in the M-fraction (RSA 1.79) (recovery 108%); other marker enzyme and protein recoveries varied between 70% to 102%. The TPP measured in the N-fraction is partially due to unbroken and damaged cells, which are pelleted together with nuclei.

Figure 3

TPP distribution in a light mitochondrial fraction after Nycodenz gradient centrifugation. The light mitochondrial fractions obtained from rat liver were subjected to Nycodenz gradient centrifugation. Fractions were collected starting from the bottom. In a first experiment (panel A), TPP content (black line) and catalase activity (marker enzyme for the peroxisomal matrix; white bars) were measured. Recoveries for TPP content and catalase activity were 60% and 70%, respectively. Thiamine, which is also present in the L-fraction, was recovered in the top fractions (data not shown). The insert shows the density (g/ml) of the gradient. In a second experiment (panels B-D), HACL1 activity was measured (panel B; white bars; recovery 78%) revealing the highest activity (35 mU/mg protein) in fraction 6, corresponding to the TPP peak (180 pmol/mg protein, black line). In panels C and D, the distribution of LDH (marker for cytosol; recovery 121%) and urate oxidase (marker for peroxisomal core; recovery 66%) is presented (white bars).

Figure 1

Reversed phase separation of thiochrome derivatives of thiamine and thiamine phosphate esters. Panel A. A mixture of derivatized standards (50 pmol each) was injected on a C18 amide column as described in Materials and Methods. The vitamins eluted in the following order: TPP (6.4 min), TMP (7.2 min) and thiamine (10.4 min). Panel B. Chromatogram of the thiochromes generated from a peroxisome enriched fraction (3.1 mg protein/ml) obtained from the Nycodenz gradient (black line), documenting the presence of TPP. Some peaks apparently originate from Nycodenz, as revealed by comparison with the profiles of the corresponding blank Nycodenz fractions, both derivatized (red line) and not derivatized (blue line).