Partial purification and characterization of two thiol proteases from hog thyroid lysosomes

Two proteases were purified from lysosomal supernatants of hog thyroids. The first step of the purification by diethylaminoethyl cellulose chromatography separated the two proteases, which were shown to have quite similar properties with respect to 2-mercaptoethanol requirement and pH optima for benzoyl-L-arginine-2-naphthylamide (BANA) hydrolytic activity, and inhibition by sulfhydryl inhibitors. One of the proteases, designated as thiol protease-1 (TP-1), was further purified by gel filtration (Sephacryl S-200) and vigorously hydrolyzed BANA but not casein. BANA hydrolytic activity of TP-1 was 50% inhibited by 10(-5) M leupeptin and by 10(-7) M L-trans-epoxysuccinyl-leucylamido(4-amino)butane. A highly purified preparation of the other protease, designated as thiol protease-2 (TP-2), was obtained after gel filtration on Sephacryl S-200 and carboxymethyl cellulose chromatography. In contrast to TP-1, TP-2 exhibited high hydrolytic activity against both casein and BANA, and was more sensitive to leupeptin and L-trans-epoxysuccinyl-leucylamido(4-amino)butane, the concentrations for 50% inhibition being 10(-7) and 10(-8) M, respectively. Electrophoresis and chromatofocusing revealed that the two proteases had different isoelectric points. TP-1 could bind to concanavalin A Sepharose and hydrolyzed L-arginine-2-naphthylamide but not carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide whereas TP-2 did not bind to concanavalin A Sepharose and hydrolyzed carbobenzoxy-L-arginyl-L-arginine methylcoumarylamide but not L-arginine-2-naphthylamide. These results suggested that TP-1 and TP-2 had properties similar to those of the liver lysosomal thiol proteases, cathepsins H and B, respectively.     

Physiological role of alpha-melanocyte-stimulating hormone in modulating the secretion of prolactin and luteinizing hormone in the female rat.

Long-term ovariectomized (OVX) rats were injected in the third cerebral ventricle with 5 microliter of the globulin fraction of an antiserum raised against alpha-melanocyte-stimulating hormone (alpha-MSH) or an equal volume of the globulin fraction of normal rabbit serum (NRS). Immunoneutralization of brain alpha-MSH produced an increase in the area under the secretion curve of prolactin (Prl), the amplitude of Prl pulses, and mean plasma Prl (P less than 0.01). In animals that had received two injections of NRS or anti-MSH and were subjected to a 2-min ether stress, Prl levels significantly increased within 5 minutes in the NRS-injected rats, whereas Prl levels in the antiserum-injected rats did not increase any further from the initially high baseline levels. The administration of antibodies against alpha-MSH produced a small increase (P less than 0.05) in the area under the secretion of luteinizing hormone (LH) and mean plasma LH; however, the number of LH pulses was unaffected. We conclude that endogenous alpha-MSH of central origin is a physiological neuromodulator of release of Prl and LH in the OVX rat and is involved in the stress-induced release of Prl.     

Lysosomal digestion of thyroglobulin: role of cathepsin D and thiol proteases

Purified hog thyroid lysosomes, prepared by a procedure previously developed in this laboratory, were used to study lysosomal digestion of [131I]thyroglobulin [131I]Tg). The lysosomal proteases were solubilized with 0.1% Triton X-100. Rates of proteolytic digestion, measured by the release of ethanol-ammonium acetate-extractable 131I, were greatly stimulated by thiol reagents. The pH optimum was also affected by the presence of thiols. In the absence of a thiol reagent, a broad pH optimum was observed, ranging from 3.5-4.5. However, in the presence of 1 mM mercaptoethanol, the maximum rate of digestion occurred at pH 5.0, very close to reported values for the internal pH of lysosomes. Pepstatin, an inhibitor of cathepsin D, markedly inhibited lysosomal digestion of [131I]Tg at concentrations as low as 0.01 micrograms/ml. Its inhibitory effect was greater at pH 3.5 (pH optimum of cathepsin D) than at pH 5.0. Leupeptin, an inhibitor of thiol proteases, was not as potent as pepstatin, but it was significantly inhibitory at a concentration of 1 microgram/ml. In contrast to pepstatin, leupeptin displayed a greater inhibitory effect at pH 5.0 than at pH 3.5. The pH optimum of hog thiol proteases has been reported to range from 5.5-6.5. The effects of the two inhibitors were additive at pH 5.0. We conclude from these results that both cathepsin D and thiol proteases play a role in lysosomal digestion of Tg. Cathepsin D appears to be quantitatively more important than thiol protease in the initial phase of the digestion. The stimulatory effect of thiols on lysosomal digestion of [131I]Tg probably involves two separate effects: 1) stimulation of thiol proteases, and 2) reduction of S-S bonds in Tg, making the protein more susceptible to attack by proteolytic enzymes. Poorly iodinated [131I]Tg was more rapidly hydrolyzed than well iodinated [131I]Tg, based on the release of ethanol-ammonium acetate-extractable 131I. However, there was little or no difference in the rate of total peptide bond cleavage between poorly iodinated and well iodinated Tg. These results suggest that the first sites of iodination of Tg are preferentially attacked by lysosomal proteases. Long term (24-h) digestion of [131I]Tg with solubilized thyroid lysosomes at pH 5.0 in the presence of thiol compounds was just as effective as digestion with pronase at pH 8.0 in liberating free 131I-labeled iodothyronines and 131I-labeled iodotyrosines. Thus, thyroid lysosomes contain the full complement of proteases and peptidases required for cleaving free iodoamino acids from Tg.     

Physiological control of growth hormone secretion by thyrotrophin-releasing hormone in the domestic fowl

Immature cockerels (4- to 5-weeks old) were passively immunized, with antiserum raised in sheep, against thyrotrophin-releasing hormone (TRH). The administration of TRH antiserum (anti-TRH) at doses of 0.5, 1.0 or 2.0 ml/kg lowered, within 1 h, the basal concentration of plasma GH for at least 24 h. The administration of normal sheep serum had no significant effect on the GH concentration in control birds. Although the GH response to TRH (1.0 or 10.0 micrograms/kg) was not impaired in birds treated 1 h previously with anti-TRH, prior incubation (at 39 degrees C for 1 h) of TRH (20 micrograms/ml) with an equal volume of anti-TRH completely suppressed the stimulatory effect of TRH (10 micrograms/kg) on GH secretion in vivo. These results suggest that TRH is physiologically involved in the hypothalamic control of GH secretion in the domestic fowl.     

Effect of calmodulin inhibitors on thyroid hormone secretion

The effect of calmodulin inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine, on TSH-induced thyroid hormone secretion from rat thyroid was examined in vivo and in vitro. The ip administration of 5 mg W-7 to the rat inhibited T4 and T3 secretion from rat thyroids at 2, 3, and 4 h after the ip injection of 2 IU TSH, and so did the ip injection of trifluoperazine at 3 and 4 h. However, the ip injection of N-(6-aminohexyl)-1-naphthalene sulfonamide as a control substance did not show any significant inhibition of T4 and T3 release. To identify the site of action of calmodulin, the effect of W-7 on (Bu)2cAMP-induced thyroid hormone secretion was tested in vitro. One hundred micromolar W-7 completely inhibited T4 release from the rat thyroid when it was enhanced by TSH or (Bu)2cAMP, suggesting that the inhibitory effect of W-7 is subsequent to cAMP formation. These results suggest that calmodulin may play a role in thyroid hormone secretion from the thyroid, acting beyond cAMP formation.     

Physiological growth hormone secretion in adult growth hormone deficiency: comparison with normal controls

A comparison was made between the pulsatile pattern of growth hormone secretion in 14 growth hormone deficient adults (serum growth hormone less than 7 mU/I following insulin-stimulated hypoglycaemia) and 14 age and sex matched controls. The 24-h secretory profile was generated by 2-hourly sampling from 0800 to 2200 h, and 30-min sampling from 2200 to 0600 h. Plasma GH in each sample was measured using a double antibody radioimmunoassay. The profiles were analysed using a computer program (Pulsar). Sleep-electroencephalography was recorded in all subjects. The total amount of GH secreted in a 24-h period (area under the curve over baseline) was significantly less in the growth hormone deficient group (P less than 0.002). The pulse frequency, amplitude, height and percentage GH secreted in peaks were also significantly less in the growth hormone deficient group (P less than 0.002 respectively). We conclude that adults deficient in GH, as defined by conventional pharmacological stimuli, are in addition physiologically deficient of pulsatile GH secretion.     

Beta2-adrenergic stimulation of thyroid hormone secretion.

The influence on thyroid hormone secretion of the nonselective beta-adrenergic stimulant isoproterenol (IPNE), of a selective beta1-adrenergic stimulant, 1-isopropylamino-3-(2-thiazoloxy)-2-propanol (ITP), and of a selective beta2-adrenergic stimulant, terbutaline, was investigated in mice. A combination of light microscopy (colloid droplet formation) and bioassay (blood radioiodine--BRI--measurements) was used. IPNE and terbutaline induced formation of colloid droplets and increased BRI levels, whereas ITP was ineffective. The responses to IPNE and terbutaline were abolished or reduced by pretreatment with L-propranolol, but were not inhibited for pretreatment with D-propranolol or phentolamine. The results indicate that secretion of thyroid hormone can be induced through the mediation of beta2-adrenergic receptors.     

The stimulation of thyroid hormone secretion in vitro by thyroid-stimulating antibodies

A new simple technique is described for the in vitro assay of thyroid-stimulating antibody (TSAb) in serum. Thyroid slices are incubated with serum in two-compartmental dialysis pot, followed by measurement of the thyroid hormone concentration in the dialysis fluid by RIA. Heat-inactivated normal serum applied directly to the tissue has no effect on the basal level of response. A dose-dependent relationship is demonstrated for both TSH and TSAb. Serum from patients with untreated Graves' hyperthyroidism is clearly shown to stimulate thyroid hormone secretion. Porcine thyroid tissue has been used and responds well to TSAb. This technique not only has the advantage of simple methodology, but also reflects the biological activity of TSAb. Therefore it will prove useful for further studies of the significance of TSAb and the pathogenesis of Graves' disease.     

The role of thyroid hormone autoantibodies in serum transport

Eleven sera known to contain thyroid hormone autoantibodies were analysed by reverse-flow electrophoresis for the equilibrium distribution of thyroid hormones between these autoantibodies and the three normal binding proteins found in serum. The binding properties of the autoantibodies determined in vitro did not necessarily predict their contribution to transport in serum of T1 and T3. Some could both bind in vitro and transport in serum. Others were able to bind both hormones but transported only one. However, some autoantibodies could be specific, binding and transporting one hormone only. In some sera, the autoantibody was the dominant transport protein having drawn hormone from thyroxine-binding globulin which is normally the most important. The autoantibodies were not saturated even in euthyroid individuals, indicating that they bind hormone reversibly and are a part of an equilibrium system.     

The role of thyronine in thyroid hormone metabolism.

Thryonine (T0) has been identified in human urine using gas chromatography-mass fragmentography (G.C.M.F.). In 22 normal individuals urinary T0 concentration was found to range between 8--25 nmol/24h. Assuming the mean normal thyroxine (T4) production rate to be approximately 100 nmol/24h, our findings indicate that less than 20% of this could be accounted for as urinary T0 excretion, thus supporting earlier findings that the peripheral metabolism of T4 is not limited solely to deiodination.