Abstract
Mode of existence of synthetic human gastrin-I (SHG-I, I. C. I.) is studied on some animal plasma both in vivo and in vitro. Radioactivity of 131I-labelled SHG-I loaded rat plasma is found in the TCA-precipitate of the plasma. By separation with Sephadex G-100 and G-50 gel column, two peaks are observed: the first is near the protein fractions of lower molecular weights, the second is at those of free SHG-I. The same phenomena are observed on 131I-SHG loaded human plasma in vivo. With Sephadex G-50 gel column fractions of SHG-I loaded human plasma in vivo, immunological and biological activities are demonstrated both on protein fractions and on those of free gastrin.
Sephadex G-100 and G-50 gel chromatography of the incubates of 131I-SHG and human plasma in vitro revealed two peaks of radioactivity: on the protein fractions and on those of free gastrin, both of which keep their antigenicity as gastrin. Disc electrophoresis and sucrose density gradient centrifugation are used to study on those protein fractions with radioactivity, and both revealed one peak on radioactivity near albumin.
Moreover, radioactivity in the protein fractions can easily be migrated to the precipitate, after incubation with human albumin specific antiserum. Then, 4% of some animal albumins are used to detect their binding abilities which are found to be as high as with their whole plasma proteins; 1×104c. p.m. of 131I-SHG is incubated with 0.5 ml of plasma or with the same volume of protein solutions at 37° for 60 minutes, at pH 7.4, protein fractions are collected with Sephadex G-50 gel column chromatography, and their radioactivity is counted. Binding ratio by B/T% are: human plasma, 12.9 to 10.6; rat plasma, 18.7; dog plasma, 20.1; rabbit plasma, 17.7; guinea pig plasma, 12.8 ; and 4% bovine serum albumin (Cohn's fraction V), 23.6; 4% rat albumin, 40.0; 4% human albumin, 13.8. On the contrary, they are very little capable of binding with the following proteins: egg albumin, 0.1; human serum gamma globulin, 2.3 and bovine serum gamma globulin, 1.4.
Such bindings are easily cleaved by incubation with 0.05 N NaOH or with 0.1 N HCl or through boiling at 100°, but incubation at 56° for 40 minutes, or overnight storage at 4° or a lower temperature show no effect on this binding. The optimal condition for in vitro binding is: pH 7.0 to 8.5, at 37° for about 120 minutes, with 4% of protein under shaking. More than 60% of the bound form is cleaved by Disc electrophoresis. A competitive inhibition in binding is observed between labelled and non labelled SHG-I of graded dose.
Conclusion: Thus, synthetic human gastrin, both 131I-labelled and non-labelled, is bound to plasma albumin of some mammals in vivo and in vitro. Those bound and free forms of gastrin maintain their immunological and biological abilities as native gastrin. The binding rate differs by species of animals.
As we have no data on endogenous gastrins or extracted ones, it is impossible to discuss about the physiological meaning of our observations, although it is interesting that the logarithmic curve on the disappearance rate of the bound form in plasma in vivo seems to be enough correlated with that of their biological activity.
