Juan M. Dellacha

Influence of some chemical treatments on the immunological properties of various mammalian growth hormones

Abstract The antigenic reactivity of some mammalian growth hormones in their native, oxidized, reduced and nitrated conditions were studied in a hemagglutination system using antisera elicited in rabbits injected with the native hormones. With the limitation imposed by the exclusive use of rabbit antisera, the following conclusions were arrived at: (a) the determinant areas are of small size in all cases; (b) the modification of the cystine, methionine, tryptophan and tyrosine residues does not greatly modify the immunological reactivity of human and horse growth hormones; (c) bovine growth hormone is very sensitive to any modification of the amino acids mentioned before with the only exception of the tyrosines.

Rate of concentration and digestion of radioactive growth hormone preparations injected in rats, as measured by the amount and nature of radioactivity in the tissues.

SummaryThe distribution of radioactivity in the tissues of the rat has been established after the administration of radioactive bovine growth hormone preparations.Bovine growth hormone was used either transformed in to a14C-guanidinated derivative, which was fully active, of labeled with less than 1 mole per mole of125I.The tissue radioactivity distribution curves obtained belong to two different categories: in kidney, liver and spleen there is an early concentration which attains a maximum in 15 minutes after the injection of the hormone, and rapidly declines. In heart, skeletal muscle, pancreas, intestine, bone and fat, the radioactivity increases gradually and a steady-state is reached after 30 to 60 minutes.Kidney is the organ where the highest concentration of radioactivity occurs. However, muscle accumulates more than 60% of the initial doses after 2 hours. Very little radioactivity appears in the urine, in this period.Similar results have been obtained with pharmacological or physiological doses of the labeled hormones.Blood plasma does not degrade the injected hormone but kidney, liver and muscle rapidly produce radioactive fragments soluble in 10% trichloro-acetic acid.

In vivo specific uptake of labeled insulin by liver, adipose tissue, pituitary, and adrenals in the turtle Chrysemys dorbigni

Insulin labeled with 125I was injected into turtles (Chrysemys dorbigni) to study its specific uptake by tissues. The maximum specific uptake of radioactivity by turtle tissues was obtained 1 hr after administration of [125I]iodoinsulin. Besides liver and adipose tissue, specific uptake of labeled insulin was detected in some endocrine glands, such as pituitary and adrenals. Both glands were as active in concentrating labeled insulin as liver and adipose tissue. A significant reduction of the uptake was observed when unlabeled insulin was injected together with the labeled hormone. This reduction was dose dependent, and the concentration of unlabeled insulin that prevented 50% of the tissue uptake of [125I]iodoinsulin was of 1 to 10 μg/kg body weight. These doses were able to induce blood glucose decrease in the turtle. Prolactin, growth hormone, or glucagon were unable to displace labeled insulin uptake. The major proportion of the radioactive material extracted from liver and pituitary 1 hr after [125I]iodoinsulin injection into turtle coeluted with [125I]iodoinsulin in Sephadex G-50 column. The presence of radioactive degradation products are consistent with the intracellular receptor mediated degradation hypothesis. These findings suggest the presence of specific insulin binding sites in liver, adipose tissue, pituitary, and adrenal glands from turtles.

Distribution and specific binding “in vivo” of iodinated growth hormones in the turtle Chrysemys dorbigni

Abstract Bovine (bGH) and human growth hormone (hGH) labeled with 125 I were injected into turtle Chrysemys dorbigni , in order to study their distributions in tissues. The radioactivity was basically concentrated by the liver and kidney, reaching a maximum 4 hr after the labeled hormone injection. Only the liver showed a significant reduction of radioactivity uptake, when labeled growth hormone was injected together with an excess of unlabeled hormone. This reduction was dose dependent. Injection of [ 125 I]iodo-hGH produced higher uptake of radioactivity by the liver than [ 125 I]iodo-bGH. The study performed suggests: (1) specific uptake of hGH or bGH by the liver; and (2) the presence in the liver of both somatogenic and lactogenic binding sites.

H-exchange behaviour and extent of reversible conformation changes in human, bovine, ovine, porcine and equine growth hormones.

Abstract The hydrogen exchange of highly purified preparations of human, bovine, ovine, porcine and equine growth hormones has been measured in various experimental conditions. All the hormones showed similar exchange behaviour. The kinetic classes of hydrogens in human, bovine, ovine and porcine growth hormones have been calculated. The extent of reversible conformation changes induced by pH variations or by urea was measured by the size of the new classes of very slow hydrogens appearing under these conditions. The greatest change was detected in human growth hormone. At physiological pH this protein was approx. 2–10 times more permeable to the solvent than all the other hormones tested.

In vivo effect of temperature on the specific liver uptake and renal degradation of labeled human growth hormone in the turtle Chrysemys dorbigni.

Abstract Turtles as heterothermic animals can be used as a biological model for studies on the influence of body temperature upon hormone binding in vivo . The time required to reach the maximum liver uptake of radioactivity following a single intraaortic injection of [ 125 I ]iodo-hGH was dependent on the body temperature. The amount of radioactivity concentrated by this organ was independent of body temperature. Breakdown of injected [ 125 I ]iodo-hGH was measured in serum at different intervals of time in turtles maintained at 14, 23, and 30°. The rate of degradation of the labeled hormone measured as TCA-soluble fragments was dependent on the body temperature. [ 125 I ]Iodo-hGH was not degraded when the labeled hormone was injected into turtles subjected to renal vessels ligation.

Specific uptake, dissociation, and degradation of 125I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands

Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na125I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na125I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125I-hGH or 125I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested.

Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells

SummaryThe effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min.1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with Ki values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a Ki = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 µM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 µM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.