Stearylamine permeabilizes the lysosomal membrane to cystine and sialic acid

Cystine efflux from isolated rat liver lysosomes was enhanced by concentrations of stearylamine that were above the critical micellar concentration. Lysosomal latency, pH, and activity of the proton-translocating ATPase were largely unaffected under controlled experimental conditions. Loss of lysosomal latency was observed at higher stearylamine to protein ratios consistent with a detergent-like mechanism of action. Partially purified cultured fibroblast lysosomes with either defective cystine or sialic acid transport lost their stored material upon exposure to stearylamine. Concentrations of stearylamine which were effective for lysosomal efflux were highly toxic for cultured fibroblasts, thus limiting its use. Under specific conditions, stearylamine apparently selectively permeabilizes the lysosomal membrane. A similar acting, but less toxic agent may be of use in the treatment of lysosomal transport disorders.     

The reduction of cystine during its transport into rat jejunal everted segments and sacs

1. Everted segments and sacs of rat jejunum were incubated in buffer containing [(35)S]cystine. 2. Concentration gradients were achieved by both segments and sacs, and the effects of duration of incubation and of cystine concentration on the isotope distribution ratios were determined. 3. Kinetic constants were determined for the uptake of cystine by both segments and sacs, and the differences between the two systems are discussed. 4. Reduction to cysteine was virtually complete intracellularly and in the sac lumen. Extensive reduction in the medium occurred only when segments were incubated. 5. Anaerobiosis prevented a concentration gradient being obtained between the medium and the tissue, but had little effect on the extent of reduction to cysteine in the tissue and sac lumen. 6. It is concluded that cystine is transported by an active process into rat jejunum, where it is present almost entirely in the reduced form, and that efflux of cysteine occurs through the serosal surface.     

The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis

The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies; the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications.     

Amino acid sequences around the cystine residues in horse growth hormone

The cystine-containing peptides of horse growth hormone were isolated and their amino acid sequences determined. Four unique half-cystine residues occur in two peptides, one containing 11 and the other, at the C-terminus of the protein, 15 amino acids. These sequences are compared with published data on growth hormones from other species.