Inactivation of Luteinizing Hormone Releasing Hormone by Rat Hypothalamic L-Cystine Arylamidase

Abstract

The rate of hydrolysis of several aminoacyl-4-nitroanilides by rat hypothalamic arylamidases was investigated. The activity of these enzymes which were mainly found in the 105 000 \m=x\ g supernatant fraction of homogenates of the hypothalamus and other parts of the brain was shown to depend upon the presence of metal ions and free thiol groups, and to be inhibited by puromycin. As previous investigations had shown that Cys\ x=r eq-\ NA is an appropriate substrate for measuring hormone effects on hypothalamic arylamidases, L-cystine arylamidase and its interaction with various peptide hormones were examined in detail. It could conclusively be shown that this enzyme interacts particularly with oligopeptides. Its activity was competitively inhibited by TRF, oxytocin, lysine vasopressin, and LH-RH. It was also shown that the biological activity of LH-RH and its inhibitory effect on the hydrolysis of L-cystine-bis-p-nitroanilide decreased when it was incubated for various periods of time with the 105 000 \ m=x\ g supernatant of rat hypothalamus homogenate.After it had been shown that LH-RH is a small peptide consisting of 10 amino acids (Schally et al 1971), it became obvious that proteolytic enzymes of the hypothalamus could play a role in the release and/or metabolic degradation of this neurohormone. The degradation of synthetic LH-RH by brain tissue was Presented in part at the

Enzyme Kinetic Studies and Inhibition by Oligopeptides of LH-RH Degradation in Rat Hypothalamus and Pituitary

“…Similarly, 48 /¿g LH-RH are degraded by an enzyme preparation of one single PIT under the same experimental conditions. This is, however, in agreement with the previous results of Kuhl and Taubert [1975a], who ob served that only 25 /<g of biologically active LH-RH remained after 1 h when 59 /rg LH-RH were incubated with the supernatant of Zi of a rat HYP. The high degrading capacity of HYP peptidases was previously observed by Marks and Stern [1974], and by Koch et al [1974], who showed that the supernatant of one rat HYP inactivated 1 /zg LH-RH within 5 min.…”

“…In view of the previous extensive use [ Kuhl and Taubert, 1975a;1975b;Kuhl el al., 1976;1977;1978] grading enzymes (arylamidases), the type of inhibition was investigated. The inhibitory ef fect of concentrations of 0-100 fiM Cys-NA on the degradation of LH-RH (5,10 or 20 /jM) by HYP and PIT enzyme preparation during 30 min of incubation is shown in figure 3.…”

“…pothalamus (HYP) and pituitary (PIT) by several investigators utilizing biological [Sundow et al, 1973;Griffiths et al, 1973;Koch et al, 1974;Kuhl and Taubert, 1975a], radioimmunological [Griffiths et al, 1974b;Kochman et al, 1975], and chemical methods [Marks and Stern, 1974]. The primary site of cleavage by these enzymes appears to be mainly the Gly6-Leu7 bond, and to a lesser degree the Pro9-(Gly-NH2)10 bond [Marks and Stern, 1974;Koch et al, 1974], The en zymes inactivating LH-RH are, however, re latively nonspecific as they are capable of de grading peptides other than LH-RH.…”

“…Marks [1976] proposed the designation of this endopeptidase as cathepsin M or neutral pro teinase. The term L-cystine arylamidase was, however, favored by Kuhl and Taubert [1975a] as the activity of the LH-RH degrading en zyme in the HYP can be measured utilizing the synthetic substrate L-cystine-bis-(4-nitroanilide) (Cys-NA). It could be shown by this expedient and relatively precise method that the enzyme system in the rat HYP and PIT can be activated in vivo by the injection of luteinizing hormone (LH), sex steroids and prostaglandins [Kuhl and Taubert, 1975b;Kuhl et al, 1976;Kuhl et al, 1977, Kuhl et al, 1978, Although these findings imply a certain involvement of the LH-RH degrading enzyme in the feedback regulation of gonadotropin release, there is as yet no definite evidence that LH-RH and Cys-NA are cleaved by the same enzyme.…”

Hypothalamic, pituitary and gonadal hormone production in relation to nutrition in the male hyrax (Procavia capensis)

“…Individual hypothalami were homogenized in 2 ml phosphate-buffered saline and then heated for 5 min in a boiling water bath to inactivate endogenous peptidases known to degrade LH-RH (Griffiths, Hooper & Hopkinson, 1973;Kuhl & Taubert, 1975;Gradwell, Millar & Symington, 1976). After centrifugation and appropriate dilution of the supernatant, LH-RH content was determined by radioimmunoassay with a specific antiserum raised to LH-RH conjugated to keyholelimpet haemocyanin and 125I-labelled LH-RH (Hendricks, Millar & Pimstone, 1975).…”