Ca-dependent inactivation of acetylcholine receptors by an endogenous transglutaminase

The nicotinic acetylcholine receptor (nAChR) from Torpedo californica and T. marmorata electric tissue polymerises irreversibly when DTE and Ca²⁺ are added to receptor-rich membranes. The polymerisation is time-dependent and complete within 3 h at 30°C. It can be completely prevented by EGTA or the transglutaminase inhibitor cystamine. Transglutaminase activity can also be monitored with the exogenous substrates [³H]putrescine and dimethylcasein. This assay can also be inhibited by EGTA or cystamine.     

Calcium dependent reversible inactivation of erythrocyte transglutaminase by acrylamide

Purified transglutaminase from human erythrocytes was shown to undergo time dependent inactivation by acrylamide added at millimolar concentration: this effect was wholly dependent on calcium ions and some protection was produced by glutamine substrates and GTP. The enzyme activity was recovered by addition of thiol based reducing agents, while ascorbic acid and sodium borohydrate were ineffective. These data are suggestive of an active site directed action of acrylamide.     

Substance P as a transglutaminase substrate: identification of the reaction products by fast atom bombardment mass spectrometry

Substance P was found to be an effective acyl donor substrate of transglutaminase in vitro, the reaction products having been examined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast atom bombardment mass spectrometry. Electrophoretic experiments showed that Substance P incorporated 14C-labeled polyamines when incubated with purified guinea pig liver transglutaminase and Ca2+. Extensive use of fast atom bombardment mass spectrometry allowed to establish that: i) a 1:1 adduct Substance P-spermine is formed; ii) only a single glutamine residue out of two, i.e. Gln-5, acts as acyl donor, iii) the single lysine residue of the neuropeptide is unable to act as acyl acceptor. A direct analytical methodology to detect transglutaminase reaction products is described.     

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in vitro inactivation.

The oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase (ATase) is demonstrated in cell extracts of Bacillus subtilis. The rate of inactivation of ATase in vitro is apparently first order with respect to oxygen concentration and ATase activity. ATase inactivation in vitro (or in vivo) cannot be reactivated by a variety of reductants. ATase is significantly stabilized to oxygen-dependent inactivation in vitro in the presence of tetrasodium phosphoribosylpyrophosphate and glutamine together. The effects of the end product inhibitors, adenosine 5-monophosphate (AMP) and guanosine 5-monophosphate (GMP), on the stability of ATase are antagonistic. AMP stabilizes ATase, whereas GMP destabilizes the enzyme. The stability of ATase can be manipulated over wide ranges by variations in the AMP/GM ratio. The effects of AMP and GMP on the inactivation of ATase in vitro are very specific. ATase is partially inhibited by 1,10-phenanthroline, suggesting that the enzyme contains iron (or some other chelatable metal ion). The inactivation of ATase in vitro is proposed to present a model for the reconstruction of the inactivation of ATase in stationary-phase cells of B. subtilis.     

Interaction with heparin protects tissue transglutaminase against inactivation by heating and by proteolysis

The considerable affinity of tissue transglutaminase for heparin was the basis for use of heparin-based affinity matrices for enzyme purification. Interaction of transglutaminase with heparin might mimic the physiological binding to membrane heparan sulfates, accounting for the limited but significant fraction of enzyme exposed at cell surface to crosslink ECM proteins. Exploring effects of heparin on transglutaminase activity and stability, we have noted that heparin only slightly affects activity in vitro, but the protein against heat treatment and proteolysis.     

Substance P

Substance P is a potent hypotensive peptide first noted in extracts of equine brain and intestinal tissue in 1931 that also stimulates the contraction of various intestinal tissues. Its isolation proved to be particularly difficult and was not accomplished until 1970 following the chance finding of a sialogogic factor in bovine hypothalamic extracts. Utilizing this biological property to monitor purification procedures, the active sialogogic peptide was obtained in pure form and identified as Substance P by multiple chemical and biological criteria. The amino acid sequence of Substance P is HArgProLysProGlnGlnPhePheGlyLeuMetNH₂. Synthetic Substance P has been prepared and a radioimmunoassay has been developed, thus facilitating well-defined research into its physiological roles. Much of the current research is centering on its possible role in sensory neurotransmission.     

Targeted inactivation of Gh/tissue transglutaminase II

The novel G-protein, G(h)/tissue transglutaminase (TGase II), has both guanosine triphosphatase and Ca(2+)-activated transglutaminase activity and has been implicated in a number of processes including signal transduction, apoptosis, bone ossification, wound healing, and cell adhesion and spreading. To determine the role of G(h) in vivo, the Cre/loxP site-specific recombinase system was used to develop a mouse line in which its expression was ubiquitously inactivated. Despite the absence of G(h) expression and a lack of intracellular TGase activity that was not compensated by other TGases, the Tgm2(-/-) mice were viable, phenotypically normal, and were born with the expected Mendelian frequency. Absence of G(h) coupling to alpha(1)-adrenergic receptor signaling in Tgm2(-/-) mice was demonstrated by the lack of agonist-stimulated [alpha-(32)P]GTP photolabeling of a 74-kDa protein in liver membranes. Annexin-V positivity observed with dexamethasone-induced apoptosis was not different in Tgm2(-/-) thymocytes compared with Tgm2(+/+) thymocytes. However, with this treatment there was a highly significant decrease in the viability (propidium iodide negativity) of Tgm2(-/-) thymocytes. Primary fibroblasts isolated from Tgm2(-/-) mice also showed decreased adherence with culture. These results indicate that G(h) may be importantly involved in stabilizing apoptotic cells before clearance, and in responses such as wound healing that require fibroblast adhesion mediated by extracellular matrix cross-linking.     

Substance P. New cyclic analogs

[Adpoc-Glu(N3)6, (Met-N3)11] substance P-(6-11)-peptide was reacted with diamines H2N(CH2) nNH2 (n = 3-10, 12) to give cyclopeptides. Subsequent careful cleavage of the Adpoc group leads to the formation of compounds of type cyclo-[H-Glu-Phe-Phe-Gly-Leu-Met-NH-(CH2) n-NH-] X HCl. The substances produce a specific two-phase myotropic effect in experiments on isolated guinea pig ileum. The compounds where n is 3, 7, 12 exhibit also a hypotensive activity when assayed on anaesthetized rats.     

Substance P and spinal neurones.

When applied by microiontophoresis, substance P (sP) had a strong, but slow and prolonged excitatory action on nearly half the neurones tested in the lumbar spinal cord of cats. Motoneuronal antidromic field potentials only occasionally showed a significant effect of sP. Cerebral cortical neurones in cats and rats were much less readily excited than spinal interneurones. Some unresponsive units showed evidence of a depressant effect of sP. Although sP may have a significant function in central afferent pathways, it is not likely to be a quickly-acting synaptic transmitter.     

Substance P enhances the proliferation of rat anterior pituitary cells in vitro

The undecapeptide substanceP(SP) was shown to be intimately involved in both the structural and functional aspects of the anterior pituitary.Yet,in addition to its influences on hormonal secretion,SP may well possess more actions in this master gland.The present study was ftherefore aimed to investigate the effect of SP on the proliferation of rat anterior pituitary cells in primary culture,It was found that SP could dose-dependently increase the incorporation of tritiated thymidine(3H-TdR) into cultured anterior pituitary cells.Other mammalian tachykinins such as neurokinin A and neurokinin B had similar effect but to varying degrees.The equipotent analogue of SP,Norleucine^11-SP(Nle^11-SP),also acted likewise.with its action antagonizable by spantide,a SP receptor blocker.To further characterize the nature of cells responsive to the challenge of SP,immunocytochemical staining against S-100 protein and some adenohypophyseal hormones was performed alone or plus autoradiography.The results showed that the percentage of S-100 proteinimmunorective cells was apparently elevated by the addtion of Nle^11-Sp for 48h,which indicates a preferential proliferation of folliculo-stellate cells under the regime .This was confirmed by increases in immunocytochemical or autoradiographical labelling indices of anterior pituitary Substance P and anterior pituitary cell proliferation.Cells treated similarly.Taken together,These results reveal that the trophic action of SP observed previously in other tissues is also present at least in cultured rat anterior pituitary cells.with responding cells being predominantly folliculo-stellate cells as typified by S-100 proteinimmunoreactivity.Therefore,an intra-pituitary trophicaction of SP in vivo could be anticipated.     

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.     

Substance P Hydrolysis by Human Serum Cholinesterase

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.     

Substance P evokes bradycardia by stimulation of postganglionic cholinergic neurons.

Substance P (SP) evokes bradycardia that is mediated by cholinergic neurons in experiments with isolated guinea pig hearts. This project investigates the negative chronotropic action of SP in vivo. Guinea pigs were anesthetized with urethane, vagotomized and artificially respired. Using this model, IV injection of SP (32 nmol/kg/50 microl saline) caused a brief decrease in heart rate (-30+/-3 beats/min from a baseline of 256+/-4 beats/min, n = 27) and a long-lasting decrease in blood pressure (-28+/-2 mmHg from baseline of 51+/-5 mmHg, n = 27). The negative chronotropic response to SP was attenuated by muscarinic receptor blockade with atropine (-29 +/- 9 beats/min before vs -8 +/- 2 beats/min after treatment, P = 0.0204, n = 5) and augmented by inhibition of cholinesterases with physostigmine (-23 +/- 6 beats/min before versus -74 +/- 20 beats/min after treatment, P = 0.0250, n = 5). Ganglion blockade with chlorisondamine did not diminish the negative chronotropic response to SP. In another series of experiments, animals were anesthetized with sodium pentobarbital or urethane and studied with or without vagotomy. Neither anesthetic nor vagotomy had a significant effect on the negative chronotropic response to SP (F3,24 = 1.97, P = 0.2198). Comparison of responses to 640 nmol/kg nitroprusside and 32 nmol/kg SP demonstrated that the bradycardic effect of SP occurs independent of vasodilation. These results suggest that SP can evoke bradycardia in vivo through stimulation of postganglionic cholinergic neurons.     

Selective inactivation of MAO-B by benzyl-dimethyl-silyl-methanamines in vitro

The alpha-silyl amines benzyl-dimethyl-silyl-methanamine and the p-fluoro and p-chloro derivatives are potent time-dependent inhibitors of rat brain MAO-B. The inhibition exhibits saturation kinetics, takes place in the enzyme active-site and is irreversible. The most potent inhibitor in the series is 4-fluorobenzyl-dimethyl-silyl-methanamine (KI = 11 microM, tau 1/2 = 2.3 min). Its selectivity for the B-form relative to the A-form of rat brain MAO is higher than 10(4). Benzyl-dimethyl-silyl-methanamines may represent a new family of anti-Parkinsonian agents.     

In vivo and in vitro induction of 'tissue' transglutaminase in rat hepatocytes by retinoic acid.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.     

Substance P receptors in mammalian central nervous system.

1. Multiple distinct affinity states or sites of substance P (SP) receptors exist in freshly-prepared rat brain membranes. 2. Substance P receptors may couple with islet-activating protein (pertussis toxin) sensitive GTP-binding protein(s). 3. Substance P receptors may be regulated Mg2+ and Na+ in an opposite manner. 4. Some important factor(s), in addition to GTP-binding protein, appear to be involved in SP binding activity. 5. An apparent molecular weight of the SP binding site is approximately 46,000 Da.     

Renal inactivation of substance P in the rat.

The activity and distribution of substance P-catabolizing enzyme(s) were studied in the rat kidney. Kidney homogenates inactive substance P 5-20 times as fast as do homogenates of intestine, liver, lung, heart or brain. The catabolizing activity was highest in the cortex and decreased progressively down the papilla. Cortex of rat kidney was homogenized and fractions enriched in microsomal membrane, final supernatant, plasma membrane, endoplasmic reticulum, brush border and intact glomeruli were prepared. The identity and homogeneity of the preparations were determined by assaying marker enzymes and by morphological examination. Substance P was catabolized most rapidly by the microsomal and plasma-membrane-enriched fractions, and least rapidly by endoplasmic reticulum or final supernatant fractions. Purified brush border of proximal tubules inactivated substance P more than 10 times as fast as isolated glomeruli. Our experiments show that substance P is catabolized at a rate that is similar to the rates of inactivation of bradykinin and angiotensin II. Further, the distribution of substance P-catabolizing activity in various kidney fractions is similar to the distribution of kininase and angiotensinase activities previously reported.     

Substance P effects on spinal nociceptive neurones.

In decerebrated spinal cats, the effects of iontophoretically applied acetylcholine (ACh) and substance P were examined on the responses of dorsal horn neurones to noxious stimulation and touch of the skin. Both agents, in amounts that did not have a significant direct effect on the neuronal firing rate, prolonged the response of the cells to noxious stimulation but did not alter that to touch stimulation. The peptide and ACh potentiated the late, but not the early, responses of dorsal horn neurones to sural A_δ and C afferent stimulation. Substance P-induced potentiation of the above responses was observed even when the agent did not produce a significant depolarization of nociceptive cells. In greater amounts, the peptide depolarized the neurones, an effect that was not associated with a detectable change in the membrane resistance. These results indicate that substance P facilitates nociceptive pathways by potentiating the subliminal fringe and, in greater amounts, by depolarizing the cells. The failure by the peptide to potentiate touch-induced excitation of the nociceptive neurones appears not to be due to the selectivity of the drug effect but due to the absence of subliminal fringe.