Comparative reactivities of 125I-secretin and 125I-minus6-tyrosyl secretin with guinea pig and rabbit anti-secretin sera.

Since secretin contains only an N-terminal histidyl and no tyrosyl residue, a synthetic secretin has been commercially prepared containing tyrosine in place of phenylalanine to facilitate the preparation of a radioiodine labeled tracer. We have found that although the rate of iodination of 6-Tyr-secretin is more rapid than that of secretin, the efficiency of iodination is not greatly increased and the shelf-life of the labeled product is not prolonged. The striking disadvantage of the use of ¹²⁵I-6-Tyr-secretin as a tracer in radioimmunoassay is its diminished immunoreactivity with several guinea pig and rabbit antisera compared to ¹²⁵I-secretin.     

Two peptidases that convert 125I-Lys-Arg(Met)enkephalin and 125I-(Met)enkephalin-Arg6, respectively,to 125I-(Met)enkephalin in bovine adrenal medullary chromaffin granules

Two peptidases which convert ¹²⁵I-Lys-Arg-ME and ¹²⁵I-ME-Arg⁶, respectively, to ¹²⁵I-ME, have been identified and characterized in bovine adrenomedullary chromaffin granules. The former is referred to as a secretory granule peptidase (SGP) and the latter as a carboxypeptidase B-like enzyme (CPB-like) [7] which is here further characterized. SGP cleaved ¹²⁵I-Lys-Arg-ME to produce only ¹²⁵I-ME and was localized in chromaffin granules which contained co²⁺-stimulated CPB-like activity, ME, and catecholamines. Both the SGP and the CPB-like enzymes appear to be thiol-metalloproteases. While the CPB-like enzyme seems likely to be involved in processing the enkephalin precursors [7], SGP may function as a trypsin-like or aminopeptidase enzyme in secretory granules.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Binding of Clostridium perfringens [125I]enterotoxin to rabbit intestinal cells

125I-Labeled enterotoxin from Clostridium perfringens was utilized to characterize the association of the enterotoxin with cells isolated from rabbit intestine and tissue homogenates from liver, kidney, and brain. The enterotoxin was found to bind in a specific and saturable manner to cells from intestine and to tissue homogenates from liver and kidney but not the brain. Detailed studies of the binding were carried out with the ileal epithelial intestinal cells. The rate and amount of binding of enterotoxin to cells appeared to be temperature dependent. Apparent affinity and association and dissociation rate constants were calculated for what appeared to be two classes of saturable binding sites. The amount of enterotoxin molecules that bound per milligram of cell protein was similar in tissue of intestinal, liver, and kidney origin (approximately 10(13) molecules/mg of cell protein). Spontaneous dissociation into the supernatant medium was observed to be much slower than expected from calculations based on the rate of association. Chaotropic ions did not enhance dissociation of the enterotoxin from cells. Enterotoxin binding was demonstrated to be heat labile (binding ability was lost after the enterotoxin was heated for 10 min at 60 degrees C). A mechanism is described whereby the enterotoxin binds and then is inserted into the membrane where it becomes trapped.     

Ganglioside Inhibition of (125 I)-Plasmin Binding to Colorectal Carcinoma Cells

Abstract

The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified gangliosides resulted in 35–60% inhibition of specific (¹²⁵ I)-plasmin binding to the cell surface. After 5 to 6 days in culture, tumor cells were pre-incubated at 4° for 1 to 4 h followed by post-incubation with (¹²⁵ I)-plasmin by techniques previously described. At 25 uM the capacity for inhibition of plasmin binding was GTlb > GQ lb ≥ GD la > GM 1 ≤ GgOse Cer. Thus a terminal sialyl moiety appears to be necessary (p < 0.05) although exogenous N-acetyl neuraminic acid was ineffective (p > 0.05), indicating a role for the lipid portion of the ganglioside. Other (glyco)lipids such as sphingosine, fucolipid H-1 and sulfatide were without significant effect. The inhibition could not be reversed by the presence of 10 mM Ca EDTA, pre-treatment of the cell with carboxypeptidase or pretreatment of plasmin with neuraminidases. The inhibition was however reversed by post-incubation in control medium without exogenous ganglioside. Cell counts determined prior to, and after ganglioside incubation showed that the effect was not due to cell death or detachment from the culture surface. The dissociation constant for (¹²⁵l)-plasmin binding was 5.6 × 10^?8 M (700,000 sites/cell), but in the presence of trisialoganglioside (GT1b), Scatchard plots suggested diversification of binding sites with 280,000 site/cell at Kd 2.6 × 10^?8 M and 820,000 sites/cell at Kd 2.1 × 10^?7 M. Another interpretation of the Scatchard plot in the presence of ganglioside was that the glycolipid imposed negative cooperativity on plasmin binding to the cell surface. These results suggest that certain gangliosides can affect tumor cell invasiveness by altering protease binding to the cell surface.     

Fragmentation of chromatin with 125I radioactive disintegrations.

The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated.