Abstract
The role of antizyme (AZ) and glycosaminoglycans in polyamine uptake by mammalian cells and mitochondria was examined using NIH3T3 and FM3A cells and rat liver mitochondria. AZ is synthesized as two isoforms (29 and 24.5 kDa) due to the existence of two initiation codon AUGs in the AZ mRNA. Most AZ existed as the 24.5-kDa form translatable from the second AUG, but a portion of the 29-kDa AZ from the first AUG was associated with mitochondria because of the presence of a mitochondrial targeting signal between the first and the second methionine. The predominance of the 24.5-kDa isoform was mainly due to the presence of spermidine and a favorable sequence context (Kozak sequence) at the second initiation codon AUG. Spermine uptake by NIH3T3 cells was inhibited by both 29- and 24.5-kDa AZs, but uptake by rat liver mitochondria was not influenced by either form of AZ. Because spermine uptake by mitochondria caused a release of cytochrome c, an enhancer of apoptosis, we looked for inhibitors of mitochondrial spermine uptake other than AZ. Cations such as Na+, K+, and Mg2+ were inhibitors of the mitochondrial uptake. It has been reported that heparan sulfate on glypican-1 plays important roles in spermine uptake by human embryonic lung fibroblasts. Heparin, but not heparan sulfate, slightly inhibited spermine uptake by FM3A cells in the absence of Mg2+ and Ca2+ but had no effect under physiological conditions in the presence of Mg2+ and Ca2+.
Highlights
The cellular content of polyamines, which play important roles in cell proliferation and differentiation, is regulated by biosynthesis, degradation, and transport [1, 2]
To study the function of both the 29- and 24.5 kDa isoforms, the nucleotide sequence around the first initiation codon was changed to an optimal Kozak sequence, and the levels of the two AZs in NIH3T3 cells were evaluated after transient transfection of the modified AZ gene
This was the case when 29-kDa AZ encoded by AZ⌬AUG2 mRNA was expressed in NIH3T3 cells
Summary
It has been reported that a polyamine uptake system exists in mitochondria [24, 25], and mitochondrial polyamines regulate calcium transport [26], ATP synthesis [27], the ATP/ADP carrier [28], and release of cytochrome c [29]. In this communication we studied in detail the role of AZ and heparan sulfate, which have been, far reported as important regulators of polyamine uptake by mammalian cells. Characteristics of polyamine uptake by mitochondria were studied from the viewpoint of inhibition of the release of cytochrome c
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