Karl Bauer

Degradation and biological inactivation of thyrotropin releasing hormone (TRH): regulation of the membrane-bound TRH-degrading enzyme from rat anterior pituitary by estrogens and thyroid hormones.

Thyrotropin releasing hormone (TRH, pyroGlu-His-Pro-NH2) is important in the regulation of adenohypophyseal hormone secretion and also serves important functions in extrahypothalamic brain areas, indicating that it is involved in neurotransmission and other forms of cellular communication. This hypothesis is strengthened by the observation that TRH is rapidly inactivated by a heterogeneously distributed ecto-enzyme which exhibits a high degree of substrate specificity. Moreover, in the rat, the activity of the membrane-bound TRH-degrading enzyme of the anterior pituitary is found to be stringently controlled by thyroid hormones and estrogens. In contrast, the activity of the TRH-degrading brain enzyme is neither influenced by thyroid hormones nor estrogens. These data indicate that the TRH-degrading brain enzyme serves specialized functions for the transmission of TRH signals and apparently represents the peptidergic equivalent to acetylcholine esterase, whereas the membrane-bound adenohypophyseal TRH-degrading enzyme itself fulfills a biologically important control function within feedback-regulatory mechanisms.

Possible role of neuropeptide degrading enzymes on thyroliberin secretion in fetal hypothalamic cultures grown in serum free medium

In the present work, we have looked for the presence of two tissular neuropeptide degrading activities, the pyroglutamate aminopeptidase (PAP) and the post-proline cleaving enzyme (PPCE), in dissociated brain cell cultures. These two activities are present in extracts of cells grown in serum-free medium and are detected at a very low level in incubation media. Depolarization of hypothalamic neurons by 60 mM K+ does not specifically increase the level of PAP and PPCE in the medium. We have also used an inhibitor of PPCE: Z-Gly-ProCHN2. This compound can be left in contact with living cells without any toxicity, and in certain conditions of incubation blocks totally and irreversibly both PAP and PPCE. This blockade results in increased levels of TRH, intracellular as well as released into the medium, spontaneously and upon K+ depolarization. These results evidence the role of degradation processes in the mechanisms regulating peptide turn-over.

Peptide uptake by astroglia-rich brain cultures.

Abstract: Uptake of carnosine has been investigated in as‐troglia‐rich primary cultures derived from brains of newborn mice. It could be demonstrated that carnosine is not degraded by these cells but rapidly taken up in an energy and sodium‐dependent process. Uptake and release of carnosine by these cells were found to be mediated by a saturable, high‐affinity transport system with apparent kinetic constants of Km=50 μMand Vmax= 22.7 nmol h1 mg protein1. Uptake of carnosine is strongly inhibited by other dipeptides as well as by various oligopeptides, e.g., Leu‐en‐kephalin. However, uptake of the radiolabeled tripeptide D Ala‐L‐Ala‐L‐Ala was not observed. Radiolabeled Leu‐en‐kephalin also did not accumulate intracellularly, even if degradation of the peptide was prevented by use of peptidase inhibitors. These results suggest that uptake of carnosine is catalyzed by a dipeptide‐specific transport system with broad substrate specificity. With neuronal cells in primary culture, uptake of carnosine or other peptides was not observed.

Chymotryptic-like hydrolysis of luliberin (LH-RF) by an adenohypophyseal enzyme of high molecular weight

An enzyme that hydrolyzes the fluorogenic chymotrypsin substrate glutaryl-Gly-Gly-Phe-β-naphthylamide has been partially purified from extracts of bovine anterior pituitaries. Like chymotrypsin, this enzyme hydrolyzes the neuropeptide Luliberin (LH-RF, <Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) at the carboxyl-side of Trp and Tyr, but it differs from the pancreatic protease by its high molecular weight, insensitivity towards OH-reactive agents and other enzymechemical parameters. It seems, however, to be identical to the “cation-sensitive neutral endopeptidase”. In the course of this study evidence has also been obtained that LH-RF is not degraded by the cystinyl-arylamidase.

Specific inhibition of post proline cleaving enzyme by benzyloxycarbonyl-Gly-Pro-diazomethyl ketone.

N-Benzyloxycarbonyl-Gly-Pro-diazomethyl ketone (Z-Gly-Pro-CHN2) was synthesized and tested as inhibitor of the post proline cleaving enzyme from bovine brain. The compound was found to inactivate the enzyme completely and irreversibly at low concentrations (0.3 microM) without affecting other proteolytic enzymes such as post proline dipeptidyl aminopeptidase, pyroglutamate aminopeptidase or trypsin. Substrates of post proline cleaving enzymes such as luliberin (LH-RH; pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and Benzyloxycarbonyl-Gly-Pro-Ala protected the enzyme from the reaction with Z-Gly-Pro-CHN2. Thus, Z-Gly-Pro-CHN2 seems to be an active site directed, specific inhibitor of post proline cleaving enzyme. When administered intraperitoneally to rats, this inhibitor (8 mg/kg) completely inactivated the post proline cleaving enzyme in all tissues studied including brain. Therefore, Z-Gly-Pro-CHN2 should be a valuable tool for studies on the physiological function of this enzyme within the metabolism of neuropeptides.

Specific fluorogenic substrates for the TRF-deamidating post proline cleaving enzyme.

The deamidation of TRF (<Glu-His-Pro-NHz) by adenohypophyseal tissue extracts is catalyzed by an enzyme which could be characterized as a post proline cleaving endopeptidase [ 1,2]. From lamb kidney an enzyme with similar chemical characteristics has been isolated [4,5]. The pituitary enzyme (M, 76 000) however, differs from the kidney enzyme (M, 115 000) by its high sensitivity towards sulfhydryl blocking agents and other properties. Both enzymes cleave proline containing peptides specifically at the Pro-X bond. The collagenase substrate Z-Gly-ProLeu-Gly-Pro [6], for example, is rapidly hydrolyzed at the Pro-Leu bond. As known, however, the Pro-X bond can also be different from a peptide bond. TRF (<Glu-Hi-Pro-NHa), TRF-alkylamides, and <Glu-His-Pro-/3-alanylamide are also hydrolyzed by these enzymes. Since the enzymatic liberation of /3-naphthylamine can easily be determined with the highly sensitive fluorometric detection method [7], Z-Hi-Pro-2NNap, Z-Gly-Pro-2-NNap and <Glu-Gly-Pro-?NNap were synthesized. Together with <Glu-HisPro-2-NNap these substances were tested as substrates of the post proline cleaving enzyme from pituitary extracts. It could be shown that Z-Gly-Pro2-NNap and Z-His-Pro-2-NNap serve as enzyme specific substrates.

Characterization of a Neutral Endopeptidase Localized in the Mitochondrial Matrix of Rat Anterior Pituitary Tissue with GnRH as a Substrate

We have determined the subcellular localization of an endopeptidase activity able to degrade gonadotropin releasing hormone (GnRH) and present in the rat adenohypophysis. After fractionation of tissue homogenates in 0.25 M sucrose by differential centrifugation, about 25% of the total cellular GnRH degrading activity was found to be sedimentable and recovered from heavy (M) and light (L) mitochondrial fractions with a distribution pattern similar to that of the mitochondrial and lysosomal reference enzymes cytochrome oxidase and beta-galactosidase. Upon further fractionation on sucrose density gradients, the activity comigrated with mitochondria. The peptidase appears endowed with a structure-linked latency; the activity is low in a freshly prepared mitochondrial fraction and increases upon treatment with membrane disrupting agents in a manner similar to that of malate dehydrogenase, a component of the mitochondrial matrix. Determination of GnRH cleavage sites was performed by amino acid analysis of the fragments obtained after incubation of the peptidase with (3H)-GnRH labelled on the pyroglutamic acid residue, in presence of carboxypeptidase and peptidyldipeptidase inhibitors. The fragments were separated by ion-exchange chromatography on an Aminex Q-15S column and purified by chromatography on silica gel plates. Fragments 1-2, 1-3, 1-4, 1-5 and 1-6 were all present as early as 1 min after the beginning of incubation. Formation of each of them was inhibited to the same extent by EDTA, mersalyl acid, dithioerythritol and Na deoxycholate. The same fragmentation pattern was observed after partial purification of the enzyme by gel filtration. These data indicate that cleavage of several peptide bonds may result from a possibly single endopeptidase located in the mitochondrial matrix space.

Degradation of LH-RH

The neuropeptide Luteinizing Hormone — Releasing Hormone (LH-RH, pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) participates in synaptic events and the hypothalamic control of adenohypophyseal hormone secretion. Since the peptide is rapidly hydrolyzed by various tissue homogenates, it has been postulated that the biological inactivation of LH-RH at the target site is catalyzed by a peptidase, which might possibly be specific for this neuropeptide. Furthermore, it has been suggested that the feedback-controlled alterations of such an enzymatic activity might be involved in the regulation of the LH-RH metabolism and thus in the control of the biological activity of this peptide (Griffiths et al., 1975; Fridkin et al., 1977; Kuhl et al., 1978; Advis et al., 1982). Alternatively, it is conceivable that LH-RH is degraded by general proteolytic enzymes, which might fulfill a scavenger function at the site of target interaction (if located in the vicinity of the receptors) or a more general metabolic clearance function at other sites. It is clear that such enzymes cannot serve a regulatory function. For answering these questions it is a prerequisite to delineate the pathway of LH-RH fragmentation and to evaluate the biochemical properties of the enzymes capable of hydrolyzing this neuropeptide.

Enzymatic Degradation of Hypothalamic Hormones at the Pituitary-Cell Level: Possible Involvement in Regulation Mechanisms

Within the concert of regulatory mechanisms balancing the functions of an organism according to the needs of the body, there are principally two control levels. On one hand, the biological effect exerted by a given concentration of an active substance is dependent on the various physiological parameters that collectively determine the “responsiveness” of the target. On the other hand, the mechanisms regulating the hormone concentrations at physiologically appropriate levels are evidently of fundamental importance, since under given physiological conditions the physiological response is, within certain limits, directly correlated with the concentration of the biologically active substance that becomes effective at the target site. Among these mechanisms, the controlled inactivation of a biologically active substance is an eminently important event. This becomes evident in the case of certain pathological disorders in which loss of control over destruction might be the primary cause of certain endocrine diseases (Knight et al., 1973).