Further studies on the inactivation of thyrotrophin releasing hormone (TRH) by enzymes in the rat hypothalamus

1980 ◽  
Vol 93 (4) ◽  
pp. 385-391 ◽  
Author(s):  
E. C. Griffiths ◽  
J. A. Kelly ◽  
N. White ◽  
S. L. Jeffcoate
Keyword(s):  
Enzyme Activity ◽  
Biologically Active ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Hypothalamic Area ◽  
Thyrotrophin Releasing Hormone ◽  
Releasing Hormone ◽  
Rat Hypothalamus ◽  
Polypeptide Antibiotic ◽  
Bacitracin A

Abstract. Thyrotrophin-releasing hormone (TRH) is known to be inactivated by enzymes present in the rat hypothalamus. To make a further study of the enzymes' action on the tripeptide, synthetic TRH was incubated with two hypothalamic subcellular fractions. By using a direct radioimmunoassay for TRH, the tripeptide was shown to be rapidly degraded by both supernatant and particulate fractions, with higher enzyme activity in the particulate fraction. Of several biologically-active peptides tested, only luteinizing hormone-releasing hormone was found to inhibit TRH inactivation; bacitracin, a polypeptide antibiotic, was also effective in inhibiting inactivation. Enzyme activity was highest in the middle hypothalamic area and lowest in the posterior hypothalamic area. Thin layer chromatography of the products of enzyme cleavage revealed the formation of only deamidated TRH in the supernatant fraction and the constituent amino acids (pyroGlu, His, ProNH2) and histidylproline-diketopiperazine by the particulate fraction, suggesting the presence of an amidase in the supernatant and two peptidases in the particulate fractions. These properties of the enzymes inactivating TRH may indicate that the enzymes could be of importance in regulating the endocrine and other functions attributed to this hypothalamic regulatory hormone.

FURTHER STUDIES ON ENZYMIC INACTIVATION OF LUTEINIZING HORMONE – RELEASING HORMONE (LH-RH) BY PEPTIDASES IN THE RAT HYPOTHALAMUS

1975 ◽  
Vol 79 (1) ◽  
pp. 7-15 ◽  
Author(s):  
E. C. Griffiths ◽  
K. C. Hooper ◽  
C. R. N. Hopkinson
Keyword(s):  
Enzyme Activity ◽  
Luteinizing Hormone ◽  
Assay System ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Rat Hypothalamus ◽  
Lh Rh ◽  
A Site

ABSTRACT Luteinizing hormone-releasing hormone (LH-RH) is known to be inactivated by peptidases in the rat hypothalamus with consequent loss of LH-releasing ability. To make a further study of the peptidases' action on the decapeptide, synthetic LH-RH and its [1–9NH2] analogue were incubated with the supernatant hypothalamic fraction containing the enzyme activity. Using an assay system measuring gonadotrophin release in ovariectomized/steroid-primed rats, both LH-RH and the [1–9NH2] analogue were found to be inactivated to different extents after incubation with the fraction, the analogue completely losing both LH- and FSH-releasing activity, and the releasing hormone almost completely losing its LH- and totally losing its FSH-releasing activity. The findings extend the initial studies by showing that the peptidases can remove both the decapeptide's intrinsic LH- and FSH-releasing activity and that these enzymes act on LH-RH at a site other than the C-terminal glycinamide, since they are able to inactivate the [1–9NH2] analogue lacking this residue.

ABSENCE OF PYROGLUTAMYL-N3im-METHYL-HISTIDYL-PROLINEAMIDE (METHYL-THYROTROPHIN RELEASING HORMONE) IN THE RAT HYPOTHALAMUS

1978 ◽  
Vol 77 (3) ◽  
pp. 405-408 ◽  
Author(s):  
A. E. PEKARY ◽  
J. E. MORLEY ◽  
J. M. HERSHMAN
Keyword(s):  
Cation Exchange ◽  
Exchange Chromatography ◽  
Mammalian Brain ◽  
Synthetic Analogue ◽  
Cation Exchange Chromatography ◽  
Thyrotrophin Releasing Hormone ◽  
Releasing Hormone ◽  
Rat Hypothalamus

Pyroglutamyl-N3im-methyl-histidyl-prolineamide (methyl-thyrotrophin releasing hormone, methyl-TRH) is a potent synthetic analogue of TRH. N3im-Methyl-histidine is present in mammalian brain and it has been suggested that methyl-TRH is a physiological releasing hormone normally present in the hypothalamus. A non-gradient cation-exchange chromatography system that uses SP-Sephadex C-25 and completely resolves methyl-TRH and TRH has been developed. Because methyl-TRH cross-reacts in the immunoassay for TRH, this assay was used to measure TRH and methyl-TRH in the chromatographic fractions. By this means it has been demonstrated that the amount of methyl-TRH present in the rat is less than 0·025 ng/hypothalamus.

THE EFFECT OF OVARIECTOMY ON THE ACTIVITY OF CERTAIN ENZYMES IN THE FEMALE RAT HYPOTHALAMUS

1972 ◽  
Vol 69 (2) ◽  
pp. 249-256 ◽  
Author(s):  
E. C. Griffiths ◽  
K. C. Hooper
Keyword(s):  
Enzyme Activity ◽  
Luteinizing Hormone ◽  
Inverse Relationship ◽  
Supernatant Fraction ◽  
Female Rat ◽  
Hormone Release ◽  
Similar Relationship ◽  
Hormone Levels ◽  
Rat Hypothalamus ◽  
Detectable Difference

ABSTRACT Previous work in the rabbit has shown that the activity of certain peptidases in the hypothalamus which inactivate oxytocin, changes with stimuli known to release gonadotrophins, and may be used as an index of gonadotrophin hormone release (Hooper 1966a,b, 1968; Frith & Hooper 1971a,b). Using this approach, a study was made of the activities of similar peptidases in the rat hypothalamus following ovariectomy, a condition known to cause gonadotrophin release. Enzyme activity in the supernatant fraction was found to decrease progressively with time after ovariectomy, until 42 days after operation, thereafter maintaining a level not significantly different from that at 42 days; there was no detectable difference in particulate enzyme activity after ovariectomy. An inverse relationship between supernatant enzyme activity and luteinizing hormone levels is suggested. It is concluded that a similar relationship to that in the rabbit exists between enzyme activity in the rat hypothalamus and the release of luteinizing hormone-releasing factor from the tissue.

THE PRESENCE OF PEPTIDASES IN THE RAT HYPOTHALAMUS INACTIVATING LUTEINIZING HORMONE RELEASING HORMONE (LH-RH)

1974 ◽  
Vol 77 (3) ◽  
pp. 435-442 ◽  
Author(s):  
E. C. Griffiths ◽  
K. C. Hooper ◽  
S. L. Jeffcoate ◽  
D. T. Holland
Keyword(s):  
Luteinizing Hormone ◽  
Reproductive Function ◽  
Assay Method ◽  
Supernatant Fraction ◽  
Peptidase Activity ◽  
Female Rat ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Rat Hypothalamus ◽  
Lh Rh

ABSTRACT The presence of peptidases in the rat hypothalamus inactivating luteinizing hormone-releasing hormone (LH-RH) has previously been demonstrated using an indirect assay method. With the development of a sensitive and specific radioimmunoassay for the releasing hormone, this technique was employed in the study of the peptidases inactivating LH-RH. It was found that supernatant fractions from both male and female rat hypothalami rapidly inactivated LH-RH, and that the peptidase activity of the supernatant fraction was higher in male than in female animals though the particulate fraction's activity was about the same in both sexes. Peptidase activity was also considerably greater in the supernatant than in the particulate fraction. These results confirm that the hypothalamus contains peptidases capable of inactivating LH-RH and give a direct indication that the enzymes may be involved in the central nervous system's control of reproductive function.

Cyclic AMP phosphodiesterase activity in mouse pancreatic islets Effects of calmodulin and phospholipids

1986 ◽  
Vol 111 (4) ◽  
pp. 533-538 ◽  
Author(s):  
Kirsten Capito ◽  
Carl Jørgen Hedeskov ◽  
Peter Thams
Keyword(s):  
Enzyme Activity ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Total Activity ◽  
Particulate Fraction ◽  
Supernatant Fraction ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Mouse Pancreatic Islets

Abstract. The activity of cyclic AMP phosphodiesterase in mouse pancreatic islets was investigated. 85% of the total activity was found in a 27 000 g supernatant fraction. The phosphodiesterase activity in the supernatant fraction, but not in the particulate fraction, was stimulated approximately 20% by Ca2+ (10−5m) and calmodulin (1 μm). The Km (cyclic AMP) of the unstimulated enzyme in the supernatant fraction was 20 μm, and the Vmax was 2 nmol/min × mg protein−1. The possible influence of a range of phospholipids was investigated. PI* and PS (150 μg/ml) inhibited the enzyme 20–30% both in the absence and presence of Ca2+/calmodulin, whereas PE, PC and PA did not affect the enzyme activity. ATP (1 mm) did not affect the particulate or supernatant fraction phosphodiesterase either in the absence or presence of Ca2+/calmodulin or Ca2+/phospholipid. It is concluded that, contrary to islet adenylate cyclase, islet cyclic AMP phosphodiesterase may be regulated by Ca2+/calmodulin.

PEPTIDASES IN THE RAT HYPOTHALAMUS INACTIVATING THYROTROPHIN-RELEASING HORMONE (TRH)

1975 ◽  
Vol 79 (1) ◽  
pp. 209-216 ◽  
Author(s):  
E. C. Griffiths ◽  
K. C. Hooper ◽  
S. L. Jeffcoate ◽  
N. White
Keyword(s):  
Anterior Pituitary ◽  
Female Rats ◽  
Peptidase Activity ◽  
Thyrotrophin Releasing Hormone ◽  
Male And Female ◽  
Releasing Hormone ◽  
Rat Hypothalamus ◽  
Male And Female Rats ◽  
Specific Radioimmunoassay ◽  
The Brain

ABSTRACT Peptidases capable of inactivating thyrotrophin-releasing hormone (TRH) have been demonstrated in the hypothalamus. With the development of a specific radioimmunoassay for TRH, this method was used to further study the enzymes acting on the releasing hormone. Whole hypothalamic homogenates from male and female rats inactivated TRH, with greater peptidase activity being found in the female animals. Separation of the homogenates into particulate (microsomal and mitochondrial) and supernatant (soluble/cytoplasmic) fractions showed approximately the same amounts of enzyme activity in both fractions, while dialysis of the fractions slightly reduced the TRH peptidase activity present, suggesting that a diffusible co-factor might be partially involved in the releasing hormone's degradation. These results confirm the presence of TRH-inactivating peptidases in the rat hypothalamus and suggest that the enzymes may be involved in some way in the mechanisms by which the brain controls thyrotrophin release by the anterior pituitary.

ALTERATIONS IN LEVELS OF THYROTROPHIN RELEASING HORMONE AND RATES OF HORMONE DEGRADATION IN HYPOTHALAMIC TISSUE OF THE DEVELOPING RAT

1980 ◽  
Vol 85 (1) ◽  
pp. 55-61 ◽  
Author(s):  
KATHLEEN PEASE ◽  
HIRAM SHEN ◽  
G. S. ACRES ◽  
J. H. RUPNOW ◽  
J. E. DIXON
Keyword(s):  
Developmental Changes ◽  
Supernatant Fraction ◽  
Neonatal Rats ◽  
Thyrotrophin Releasing Hormone ◽  
Male And Female ◽  
Releasing Hormone ◽  
Adult Level ◽  
Fetal Rats ◽  
Hypothalamic Tissue ◽  
Developing Rat

SUMMARY Developmental changes in levels of hypothalamic thyrotrophin releasing hormone (TRH) and the role that hypothalamic degrading enzymes may play in these alterations were investigated. Levels of TRH in male and female fetal rats and in male neonatal rats were measured by radioimmunoassay. The hormone content of the hypothalamus was shown to increase from less than 1 ng at 1–4 days of age to approximately 10 ng at 20 days of age. Thereafter, the content of TRH declined to the adult level of about 5 ng. The ability of fractionated hypothalamic homogenates to degrade TRH was measured over the same time. The 27 000 g supernatant fraction contained a degrading activity that yielded only radiolabelled deamido-TRH upon incubation with [l-proline 2,3-3H]TRH. The corresponding particulate fraction contained at least two distinct TRH degrading activities as determined by the number of metabolites present. Changes in rates of degradation were not large enough to account for the differences observed in levels of TRH.

THE EFFECT OF NEONATAL ANDROGEN ON THE ACTIVITY OF CERTAIN ENZYMES IN THE RAT HYPOTHALAMUS

1972 ◽  
Vol 70 (4) ◽  
pp. 767-774 ◽  
Author(s):  
E. C. Griffiths ◽  
K. C. Hooper
Keyword(s):  
Enzyme Activity ◽  
Luteinizing Hormone ◽  
Particulate Fraction ◽  
Female Rats ◽  
Testosterone Treatment ◽  
Rat Hypothalamus ◽  
Hypothalamic Control ◽  
Activity Of Enzymes ◽  
Lh Secretion

ABSTRACT The activity of enzymes inactivating oxytocin in the hypothalamus changes with stimuli known to release gonadotrophins and may be related to luteinizing hormone-releasing factor (LH-RF) release (Frith & Hooper 1971a,b; Griffiths & Hooper 1972). In the work presented here, enzyme activity was determined following neonatal testosterone treatment to assess any changes in the hypothalamic control of LH secretion. Supernatant and particulate fraction activities in female rats were depressed to male levels following injection on day 3; supernatant activity after injection on day 10 was also slightly decreased. These findings suggest that masculinization of the mechanisms controlling LH secretion in the hypothalamus, produced by neonatal testosterone, is caused by changes in LH-RF metabolism and that these peptidases in the rat hypothalamus are responsible for this metabolism.

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