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Degradation of TRH-Gly and TRH by human postmortem brain peptidases
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AN I M M U N O C Y T O C H E M I C A L STUDY USING C - T E R M I N A L L Y PANCREASTATIN ANTISERA
D I R E C T E D RAT
W.J. CURRY, C.F. JOHNSTON, C. SHAW and K.D. BUCHANAN, Medicine, The Q u e e n ' s U n i v e r s i t y of Belfast, Belfast,
D e p a r t m e n t of BTI2 6BJ.
A p p l i c a t i o n of p o r c i n e p a n c r e a s t a t i n (PST) a n t i s e r a to rat tissues have i n d i c a t e d limit8 ~ c r o s s r e a c t i v i t y . A n t i s e r a were raised to a s y n t h e t i c C - t e r m i n a l Lys L - n o n a p e p t i d e fragment of rat PST. Susa fixed wax e m b e d d e d s e c t i o n s were r e h y d r a t e d and i m m u n o s t a i n e d with a n t i s e r u m 607.1. B o i l e d 0.5M acetic acid tissue were a s s a y e d using a n t i s e r u m 609.5 and were s u b j e c t e d to S e p h a d e x G-100 chromatography. Intense s t a i n i n g o c c u r r e d in: cells t h r o u g h o u t the fundic mucosa, fewer being d e t e c t e d in antral mucosa; f l a s k - s h a p e d e n d o c r i n e cells d i s t r i b u t e d t h r o u g h o u t the small intestine; all adrenal m e d u l l a r y cells; n u m e r o u s cells of the a n t e r i o r p i t u i t a r y and in p e r i p h e r a l islet cells. Moderate IR o c c u r r e d in cells at the p e r i p h e r y of t h y r o i d follicles, while weak IR was d e t e c t e d in some cells of the islet cell core. Tissue levels (ng/g S.E. of mean (n=3)): - p a n c r e a s 0.42±0.16; fundus 8 . 6 6 ± 0 . 4 0 5 ; a n t r u m 3.16±0.21; intestine 0.48±0.02; thyroid 7.41± 0.34; adrenal 102.53± 8.05; p i t u i t a r y 210.39±11.49; brain 0.65±0.056. GI00 p r o f i l e s of the adrenal and p i t u i t a r y i n d i c a t e d a single large m o l e c u l a r w e i g h t (MW) form, while large and i n t e r m e d i a t e MW forms were d e t e c t e d in the brain, pancreas, t h y r o i d and antrum. In the fundus and intestine large, i n t e r m e d i a t e and small m o l e c u l a r forms were detected. This investigation d e m o n s t r a t e d a similar ICC and RIA d i s t r i b u t i o n of rat PST to that o b s e r v e d in pig. H o w e v e r c h r o m a t o g r a p h i c analysis has failed to detect any m o l e c u l e of similar size to p o r c i n e PST.
DEGRADATION OF TRH-GLY AND TRH BY HUMAN POSTMORTEM BRAIN PEPTIDASES
M. CUSAC~ E.C. GRIFFITHS, KIK. RAJANI and R.G. ROBERTSON l, Depts. of Physiological Sciences and Cell and Structural Biology, University of Manchester, Manchester M13 9PT Thyrotrophin-releasing hormone (TRH, Glp-His-ProNHs) is biosynthesized from a high molecular-weight precursor, via several post-translational steps including the conversion of TRH-Gly (Glp-His-Pro-GlyOH) to TRH by an m-amidase [Ann.Rev. Physiol. 50 333-344 (1988)]. The degradation of these two peptides, TRH-Gly and TRH, by peptidases present in two subcellular fractions of human postmortem cortex has been studied by HPLC [Regul.Pept. lO 145-155 (1985)]. The proline endopeptidase present in the soluble fraction was capable of converting both peptides to deamido-TRH (TRH-OH), with a similar, lesspronounced conversion in the particulate (mitochondrial/microsomal) fraction. TRH was degraded to cyclo(His-Pro) and Glp-His by pyroglutamyl aminopeptidase and imidopeptidase in the particulate fraction. For TRH-Gly, the major particulate fraction products were Glp-His and a peak with the same retention time as TRH. This peak was increased by incubation in the presence of 50mM ascorbic acid, as was that of Glp-His, though TRH-OH formation was reduced. Addition of copper ions (4mM) inhibited all product formation from TRH-Gly by both fractions, whereas addition of both copper ions and ascorbic acid together reduced TRH-GIy disappearance. These defined co-factors of the ~-amidase may perform another function in reducing the degradation of TRH-GIy by brain peptidases, thus facilitating TRH biosynthesis. [M.C. & K.K.R. were supported by the MRC]
AN I M M U N O C Y T O C H E M I C A L STUDY USING C - T E R M I N A L L Y PANCREASTATIN ANTISERA
D I R E C T E D RAT
W.J. CURRY, C.F. JOHNSTON, C. SHAW and K.D. BUCHANAN, Medicine, The Q u e e n ' s U n i v e r s i t y of Belfast, Belfast,
D e p a r t m e n t of BTI2 6BJ.
A p p l i c a t i o n of p o r c i n e p a n c r e a s t a t i n (PST) a n t i s e r a to rat tissues have i n d i c a t e d limit8 ~ c r o s s r e a c t i v i t y . A n t i s e r a were raised to a s y n t h e t i c C - t e r m i n a l Lys L - n o n a p e p t i d e fragment of rat PST. Susa fixed wax e m b e d d e d s e c t i o n s were r e h y d r a t e d and i m m u n o s t a i n e d with a n t i s e r u m 607.1. B o i l e d 0.5M acetic acid tissue were a s s a y e d using a n t i s e r u m 609.5 and were s u b j e c t e d to S e p h a d e x G-100 chromatography. Intense s t a i n i n g o c c u r r e d in: cells t h r o u g h o u t the fundic mucosa, fewer being d e t e c t e d in antral mucosa; f l a s k - s h a p e d e n d o c r i n e cells d i s t r i b u t e d t h r o u g h o u t the small intestine; all adrenal m e d u l l a r y cells; n u m e r o u s cells of the a n t e r i o r p i t u i t a r y and in p e r i p h e r a l islet cells. Moderate IR o c c u r r e d in cells at the p e r i p h e r y of t h y r o i d follicles, while weak IR was d e t e c t e d in some cells of the islet cell core. Tissue levels (ng/g S.E. of mean (n=3)): - p a n c r e a s 0.42±0.16; fundus 8 . 6 6 ± 0 . 4 0 5 ; a n t r u m 3.16±0.21; intestine 0.48±0.02; thyroid 7.41± 0.34; adrenal 102.53± 8.05; p i t u i t a r y 210.39±11.49; brain 0.65±0.056. GI00 p r o f i l e s of the adrenal and p i t u i t a r y i n d i c a t e d a single large m o l e c u l a r w e i g h t (MW) form, while large and i n t e r m e d i a t e MW forms were d e t e c t e d in the brain, pancreas, t h y r o i d and antrum. In the fundus and intestine large, i n t e r m e d i a t e and small m o l e c u l a r forms were detected. This investigation d e m o n s t r a t e d a similar ICC and RIA d i s t r i b u t i o n of rat PST to that o b s e r v e d in pig. H o w e v e r c h r o m a t o g r a p h i c analysis has failed to detect any m o l e c u l e of similar size to p o r c i n e PST.
DEGRADATION OF TRH-GLY AND TRH BY HUMAN POSTMORTEM BRAIN PEPTIDASES
M. CUSAC~ E.C. GRIFFITHS, KIK. RAJANI and R.G. ROBERTSON l, Depts. of Physiological Sciences and Cell and Structural Biology, University of Manchester, Manchester M13 9PT Thyrotrophin-releasing hormone (TRH, Glp-His-ProNHs) is biosynthesized from a high molecular-weight precursor, via several post-translational steps including the conversion of TRH-Gly (Glp-His-Pro-GlyOH) to TRH by an m-amidase [Ann.Rev. Physiol. 50 333-344 (1988)]. The degradation of these two peptides, TRH-Gly and TRH, by peptidases present in two subcellular fractions of human postmortem cortex has been studied by HPLC [Regul.Pept. lO 145-155 (1985)]. The proline endopeptidase present in the soluble fraction was capable of converting both peptides to deamido-TRH (TRH-OH), with a similar, lesspronounced conversion in the particulate (mitochondrial/microsomal) fraction. TRH was degraded to cyclo(His-Pro) and Glp-His by pyroglutamyl aminopeptidase and imidopeptidase in the particulate fraction. For TRH-Gly, the major particulate fraction products were Glp-His and a peak with the same retention time as TRH. This peak was increased by incubation in the presence of 50mM ascorbic acid, as was that of Glp-His, though TRH-OH formation was reduced. Addition of copper ions (4mM) inhibited all product formation from TRH-Gly by both fractions, whereas addition of both copper ions and ascorbic acid together reduced TRH-GIy disappearance. These defined co-factors of the ~-amidase may perform another function in reducing the degradation of TRH-GIy by brain peptidases, thus facilitating TRH biosynthesis. [M.C. & K.K.R. were supported by the MRC]