Jeffrey F. McKelvy

An Aminopeptidase from Bovine Brain Which Catalyzes the Hydrolysis of Enkephalin

An aminopeptidase from bovine brain which catalyzes the hydrolysis of the tyrosyl1‐glycine2 bond of methionine5‐enkephalin has been purified to electrophoretic homogeneity. The enzyme also catalyzes the hydrolysis of di‐peptides, tripeptides, and amino acid β‐naphthylamides. The enzyme can be inactivated by dialysis against EDTA, and reconstituted with divalent metal ions. Inhibition of the enzyme is observed in the presence of p‐chloromercuribenzoate and puromycin, the latter compound not being hydro‐lyzed by the enzyme. The enzyme is composed of a single polypeptide chain of molecular weight approx. 100,000. The properties of this enzyme are similar to those reported for other brain aminopeptidases active on enkephalin, although distinct differences are observed.

Enzymes involved in the degradation of thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) in bovine brain

As part of an investigation of neuropeptide inactivation mechanisms, we have resolved an enzymatic activity in bovine brain which catalyzes the deamidation of thyrotropin releasing hormone (TRH) and the hydrolysis of the Pro9--Gly10--NH2 bond of luteinizing hormone releasing hormone (LH-RH) from a second LH-RH degrading activity which does not degrade TRH. The former activity is similar, if not identical to, the post-proline cleaving enzyme in kidney as it is active toward the post-proline cleaving enzyme substrate CbzGly--Pro--Leu--Gly and inhibited by CbzPro--Phe and diisopropylfluorophosphate. In addition, products derived from the degradation of TRH and LH-RH by this activity show a specific cleavage on the carboxyl side of a proline residue. The latter activity has not yet been characterized with respect to its site of cleavage of the LH-RH molecule due to the presence of other contaminating peptidases.

Characterization of the site of cleavage of luteinizing hormone-releasing hormone under conditions of measurement in which LHRH degradation undergoes physiologically related change

The possibility that the degradation of luteinizing hormone-releasing hormone (LHRH) may contribute to the establishment of appropriate levels of the decapeptide neurohormone for gonadotropin release is of potential importance to this neuroendocrine regulatory pathway. We have recently provided evidence that total LHRH degradation undergoes a transient decrease in homogenates of the hypothalamic median eminence immediately prior to the luteinizing hormone surge during the first estrus cycle at puberty in the female rat. Total LHRH degradation in this situation was highly correlated with the appearance of LHRH 1–5 . In order to more fully characterize LHRH degradation under these conditions of measurement, we have carried out an analysis of all LHRH degradation products by high performance liquid chromatography/amino acid analysis. Our findings suggest that LHRH degradation occurring under these conditions is due to cleavage at the Tyr 5 -Gly 6 bond.

Presence and Ontogeny of Enkephalin and Substance P in the Chick Ciliary Ganglion

Abstract: The avian ciliary ganglion has been reported to contain both enkephalin and substance P in preganglionic terminals. However, extensive biochemical characterization of these antigens has not been completed. Using radioimmunoassays specific for Met5‐and for Leu5‐enkephalin and for substance P we identified immunoreactive substances in ganglionic extracts that comigrate on HPLC columns with standard Met5‐ and Leu5‐enkephalin and with substance P. The ontogeny of Met5‐enkephalin and substance P during embryogenesis was determined in ganglionic extracts and we found that the content of Met5‐enkephalin in the ganglion reached a peak at embryonic stage 37 whereas the content of substance P in the ganglion reached its maximum in the adult.

In vivo biosynthesis and transport of oxytocin, vasopressin and neurophysin from the hypothalamus to the spinal cord

The biosynthesis of oxytocin, vasopressin and their associated neurophysins were studied in the projection from the paraventricular nucleus of the hypothalamus to the spinal cord in individual freely-moving adult male rats. Neuropeptide biosynthesis was studied in vivo by the delivery of [35S]cysteine through stereotaxically implanted indwelling cannulae using an osmotic minipump delivery system. Following the appropriate chase times, the neural lobe and spinal cord segments T1-T4 and T12-L2 were removed from fresh tissue; in addition, the nucleus of the solitary tract was punched from frozen coronal sections. The radiolabeled peptides were purified from the tissue homogenates by sequential linear and exponential gradient elution from reverse-phase high performance liquid chromatography columns. This approach has allowed us to purify radiolabeled oxytocin and vasopressin from both the upper and lower spinal cord. However, the kinetics of oxytocin and vasopressin biosynthesis appeared to be remarkably different, as judged by their differential labeling with different pulse and chase times. Additionally, the use of different chase periods following the pulse of radiolabel has allowed us to determine that oxytocin reaches the spinal cord via the fast component of axonal transport (greater than 8 mm h-1). Using immunoprecipitation and purification by high performance liquid chromatography, we were also able to purify radiolabeled neurophysins from spinal cord tissue homogenates. These results lend further support to a role for oxytocin and vasopressin in the modulation of autonomic nervous system function and to the role of the paraventricular nucleus as an integration center for endocrine and autonomic function.

TRH in the rat cerebellum: II. Uptake by cerebellar slices

We investigated the in vitro uptake of exogenous TRH by rat cerebellar slices. TRH was found to be taken up via a process sharing many of the properties of a high affinity transport system, viz: (1) saturation kinetics; (2) high affinity kinetic constants (Km1 = 1.06 X 10(-5) M, Km2 = 5.6 X 10(-6) M); (3) temperature sensitivity (Q10 = 1.48); (4) dependency on the sodium concentration in the incubation medium; and (5) tissue/medium ratios greater than 1. After 60 min of incubation at 27 degrees C in the presence of Bacitracin, the tissue/medium ratio was 5:1. Under these conditions 70% of the total label was recovered as [3H-Pro]TRH. These findings demonstrated the existence of removal mechanisms for TRH in the cerebellum and gave further support to the hypothesis that TRH plays a functional role removal mechanisms for TRH in the cerebellum and gave further support to the hypothesis that TRH plays a functional role in this extrahypothalamic brain region.

Accumulation of Thyrotropin Releasing Hormone by Rat Hypothalamic Slices

Abstract: It has been postulated that thyrotropin releasing hormone (TRH) may play an active role in synaptic transmission. If such is the case, an inactivation mechanism must exist, in analogy to other neuroactive substances. In these studies we have considered the possibility that TRH may be taken up by rat hypothalamic slices. We observed that in the presence of bacitracin TRH was stable in the medium up to 90 min. We detected intact [3H]Pro‐TRH associated with the slices as evidenced by TLC and paper electrophoresis; the association was time‐dependent up to 60 min, and the maximum tissue‐to‐medium ratio was 1.3 at this time. At 5 min incubation, 30‐50% of the TRH was not extracellular, and the plot of TRH‐associated tissue versus the total amount of tissue was linear up to two hypothalami per flask. The association was saturable (Km 1.07 μM) and temperature‐dependent, and the saturable part of the accumulation was inhibited by ouabain, dinitrophenol, and the absence of glucose. These results suggest that an uptake mechanism for TRH exists in the hypothalamus; its physiological relevance remains to be elucidated.

TRH in the rat cerebellum: I. Distribution and concentration.

We determined the regional distribution and concentration of endogenous TRH in the rat cerebellum. Radioimmunoassay of endogenous TRH extracted and purified from five different regions of the rat cerebellum and whole hypothalamus showed that the cerebellar vermis contained 24 pg/mg, the hemispheres 74 pg/mg, the deep cerebellar nuclei 148 pg/mg, and the flocculo-nodular region 559 pg/mg of TRH. The highest concentration of TRH was in the cerebellar paraflocculi, which contained 786 pg/mg. The hypothalamic concentration of TRH was 465 pg/mg. Assay of the non-purified tissue fractions (crude extracts) resulted in lower TRH values in accordance with data previously reported by other authors. Bioassay analysis of TRH in purified fractions resulted in values similar to those obtained by radioimmunoassay. On the basis of these findings we hypothesize a functional role for TRH in the cerebellum.

[34] Methods for the study of the biosynthesis of neuroendocrine peptides in vivo and in vitro

Publisher Summary This chapter describes the techniques for studying the de novo biosynthesis of the neurohypophysial hormones oxytocin and vasopressin and their neurophysins, both to their posterior pituitary and brainstem terminal fields, and of the hypophysiotropic hormone luteinizing hormone-releasing hormone (LHRH). The chapter focuses on in vivo studies, with labeling studies, of neuroendocrine peptides and found that in vivo biosynthetic studies involving isotope administration to sites of cell bodies of origin of a peptide system of interest gives the highest levels of isotope incorporation. This approach has proved to be valuable to the study of other peptidergic systems in the central nervous system of the rat, such as the striatonigral substance P system, and the hypothalamic proopiomelanocortin system. In some cases, however, for example, to observe the directionality of the effects of feedback signals, especially steroid hormones, on neuroendocrine peptide biosynthesis, it is convenient to carry out studies in vitro . The chapter also describes the techniques for the study of luteinizing hormone-releasing hormone (LHRH) biosynthesis in primary dispersed cell cultures of perinatal rat hypothalami.

Biosynthesis of Hypothalamic Peptides

In this paper, I will attempt to summarize current knowledge about the biosynthesis of hypothalamic peptides of biological interest and will emphasize methodological aspects of peptide biosynthetic studies, a subject which I feel is of utmost importance at the present time.