Chemistry and Biology of Proteins - F. Haurowitz 1953

Proteins with Hormonal Activity
Anterior Pituitary Hormones

Although the Pituitary Gland is a remarkably small organ—the human pituitary weighs roughly 0.7 g—it houses a vast array of crucial Hormones, the majority of which reside in its anterior lobe. The Hormones of the anterior pituitary differ fundamentally in their mode of action from those of the posterior lobe. For instance, the previously mentioned Posterior Pituitary Hormones, oxytocin and pitressin, act very rapidly, eliciting a marked physiological response within minutes of injection. In contrast, Anterior Pituitary Hormones act extremely slowly; as a rule, their effects become apparent only after many hours or even days. The changes observed following the administration of many of these hormones are typically not the result of a direct action, but rather stem from their regulatory influence on various other Endocrine glands. Thus, the action of pituitary gonadotropic hormone is frequently manifested through an enhanced activity of the Gonads, whereas The Effect of thyrotropic hormone is detected As a result of increased thyroid hormone secretion. It stands to reason that the outcomes of such indirect actions cannot manifest immediately, but require a considerable span of time to develop.

It is currently believed that the minute anterior lobe of the pituitary produces 10 to 15 distinct hormones. We are able to deduce the existence of most of these substances solely by observing their specific physiological activity, as only a few have thus far been isolated as defined chemical entities. To date, only the following anterior pituitary hormones have been successfully isolated and separated from one another [65, 66]: Growth Hormone, thyrotropic hormone, adrenocorticotropic hormone, lactogenic hormone (prolactin), and two gonadotropic hormones—one stimulating follicle maturation (FSH) and the other interstitial Cells (ICSH). The Separation of most of these hormones is achieved via ammonium sulfate fractionation [67]. More recently, it has been demonstrated that the diabetogenic hormone of the pituitary [68] is identical either to growth hormone or to a mixture of growth hormone and adrenocorticotropic hormone [69, 70]. Our knowledge regarding The Nature of several anterior pituitary hormones remains rather incomplete. For example, it has not yet been elucidated whether the ketogenic hormone, the parathyrotropic hormone, and certain other factors described by various investigators are identical to any of the aforementioned hormones isolated from the anterior lobe.

Growth hormone is extracted from the pituitary gland using alkaline Solvents [71–73], such as lime Water [66]. From these extracts, it can be precipitated by The addition of 30% acetone [74], ammonium sulfate [73], sodium sulfate [75], or by adjusting the solution to pH 6.85 (its isoelectric point) [66]. Consequently, growth hormone exhibits The properties of a typical globulin; it is thermolabile, yet it is not denatured by urea [76]. Growth hormone has been obtained in crystalline form from 15% alcoholic solutions [77, 78]; its Amino Acid Composition is presented in Table 1, and its molecular weight is 49,200 [79].

Alkaline extracts of the anterior pituitary also contain thyrotropic hormone. It is more soluble than growth hormone and is not precipitated by 30% acetone [74]. Thyrotropic hormone dissolves in 50% acetone and 50% pyridine [80] and, unlike true Proteins, is not precipitated by trichloroacetic acid [81, 82]. All attempts to obtain this hormone in a pure state have proven unsuccessful, despite subjecting it to repeated repurification (up to 100 times). The Molecular Weight of this hormone is 10,000 [83]. Its best preparations are free from contamination by growth hormone and gonadotropic hormones [82]. The action of thyrotropic hormone on The Thyroid Gland is evidenced by an increase in thyroid weight [80]. Upon administration of thyrotropic hormone to an animal, the iodine content of its thyroid gland decreases while that of the Blood increases [81]. Thyrotropic hormone stimulates cellular Respiration in the thyroid, but exerts no comparable effect on the cells of other Tissues [84]. This effect is also observable in experiments utilizing thyroid tissue slices [85]. Evidently, thyrotropic hormone possesses a specific affinity for thyroid tissue and stimulates its activity. The Mechanism of this influence remains entirely unexplored to date.

Adrenotropic hormone (adrenocorticotropic hormone) of the anterior pituitary is the most stable of all anterior pituitary hormones. It is not degraded by boiling 0.25% Hydrochloric acid. The isolation of adrenocorticotropic hormone is carried out as follows: the hormone is extracted from the gland using acidified acetone, subsequently precipitated with a higher concentration of acetone, and re-extracted with a sodium phosphate solution. From this final solution, the hormone is salted out using ammonium sulfate [86, 87]. The molecular weight of this hormone is 20,000, and its isoelectric point lies at pH 4.7–4.8. The hormone contains 2.3% sulfur [66]. Its Introduction into the Organism induces hyperactivity of the adrenal cortex. Of great interest is the fact that adrenocorticotropic hormone retains its biological activity even when 50% hydrolyzed by Pepsin [87]. The biological activity of the hormone is abolished upon the substitution of its free amino and carboxyl groups or its constituent Tyrosine residue [88].

Prolactin, or the lactogenic hormone of the anterior pituitary, is a protein with a molecular weight of 22,000 and an isoelectric point at pH 7.5 [89]. Prolactin has been successfully separated from other pituitary hormones [90, 92]. It stimulates the growth not only of the mammary gland ducts, but of the glandular parenchyma itself.

Pituitary gonadotropic activity was initially attributed to the action of a single hormone. Later, however, it was discovered that gonadotropic pituitary preparations could be resolved into two hormones possessing distinct properties. One of these, precipitated by ammonium sulfate at pH 5–6, causes luteinization in the Ovaries and was therefore initially designated as luteinizing hormone (LH) [93]. Subsequently, it was given the new designation ICSH (interstitial Cell-stimulating hormone) upon the discovery that it also acts upon the interstitial Cells of the male gonad. In contrast to this factor, the second gonadotropic hormone is precipitated only at high concentrations of ammonium sulfate. This hormone stimulates follicular development in the Ovary and is termed follicle-stimulating hormone (FSH).

Gonadotropic hormones can be separated from one another by means of fractional ammonium sulfate precipitation. Another method for separating these hormones involves extracting the gland with an aqueous pyridine solution and precipitating ICSH with ethyl alcohol [94–96]. Both hormones are characterized by a high carbohydrate content and belong to the Class of typical Glycoproteins. For instance, luteinizing hormone from the porcine pituitary has a molecular weight of 100,000 and contains 4.5% mannose and 5.9% hexosamine [97]. Luteinizing hormone obtained from the ovine pituitary contains 2.8% mannose and 2.2% hexosamine; its molecular weight is 40,000, and its isoelectric point lies at pH 4.6 [98]. Pituitary follicle-stimulating hormone contains 1.2% mannose and 0.6% glucosamine [99]. The total carbohydrate content within it reaches 10–13% [100]. The molecular weight of follicle-stimulating hormone is 70,000, and its isoelectric point lies at pH 4.5 [101].

In addition to the two pituitary gonadotropic hormones described above, another gonadal stimulant has been detected in the animal organism during Pregnancy. This hormone (chorionic gonadotropin) is produced in the Placenta and holds immense clinical significance because it begins to be excreted in the urine during the very first days of gestation, thereby enabling the Diagnosis of pregnancy via the Aschheim-Zondek test [102]. It was initially believed that the urinary gonadotropin designated by Zondek as prolan was identical to the pituitary gonadotropic hormone and likely represented a mixture of luteinizing and follicle-stimulating hormones [103, 104]. Subsequently, however, this theory was discarded. The interpretation of experimental data is complicated by the circumstance that gonadotropin may act upon the ovary not directly, but rather via the pituitary gland by stimulating The production of the latter's two gonadotropic hormones [105, 106]. Gonadotropic hormone derived from urine differs from pituitary gonadotropic hormones by its remarkable heat stability [107, 108] and an even higher carbohydrate content. The view that urinary gonadotropin is indeed synthesized in the placenta is supported by observations of the production of this hormone in placental tissue cultures [109].

Gonadotropic hormone can be isolated from urine through adsorption onto tungstic acid [110], benzoic acid [111], Lloyd's reagent [112], quinine [113], uracil phosphate, or aluminum [114]. It is insoluble in both ethyl alcohol and acetone and exhibits stability toward these solvents. Consequently, it becomes feasible to dissolve the adsorbents in alcohol or acetone, thereby recovering the hormone as an insoluble precipitate. The hormone may likewise be eluted from inorganic adsorbents using weakly alkaline solutions [114]. Urinary gonadotropic hormone is also adsorbed onto charcoal, from which it is subsequently eluted with phenol [110].

Urinary gonadotropic hormone is precipitated upon saturation with ammonium sulfate, but is not precipitated by sulfosalicylic acid [115]. It is inactivated by pepsin and Trypsin, yet remains resistant to peptidases, which corroborates its proteinaceous nature [116]. The purified hormone contains 8.4% nitrogen and 18% CARBOHYDRATES [117] and is free of phosphorus and sulfur contaminants [117, 118]. The molecular weight of this hormone is 100,000; its carbohydrate moiety consists primarily of hexosamine digalactose [119].

Yet another gonadotropic hormone (chorionic gonadotropin), containing 25% carbohydrates, has been identified in the serum of pregnant mares. Its isoelectric point lies near pH 2.6, and its molecular weight is 60,000–80,000 [117]. This chorionic gonadotropin is inactivated by salivary amylase. On this basis, it is hypothesized that its hormonal activity is intimately linked to the presence of a carbohydrate component within its molecular Structure [120].