"Near Cure" treatment of severe acute EAE in MIF-1-deficient female and male mice with a bifunctional MHCII-derived molecular construct.

Our previous studies demonstrated increased serum levels of macrophage migration inhibitory factor (MIF-1) and its homologue, MIF-2, in males during MS progression; and that genetically high-MIF-expressing male subjects with relapsing multiple sclerosis (MS) had a significantly greater risk of conversion to progressive MS than lower-MIF-expressing males and females. However, female MS subjects with severe disease expressed higher levels of CD74, the common MIF-1/MIF-2 receptor, on blood cells. In the murine model of MS, experimental autoimmune encephalomyelitis (EAE), both male and female mice lacking MIF-1 and/or MIF-2 were clinically improved during development of moderately severe disease, thus implicating both homologs as co-pathogenic contributors. The current study using MIF-deficient mice with severe acute EAE revealed a highly significant reduction of EAE scores in MIF-1-deficient females, in contrast to only minor and delayed reduction of clinical signs in MIF-1-deficient males. However, clinical EAE scores and factor expression were strongly suppressed in males and further reduced in females after treatment of WT and MIF-1-, MIF-2- and MIF-1/2-DUAL-deficient female and male mice with a MHCII DRα1-MOG-35-55 molecular construct that competitively inhibits MIF-1 & MIF-2 signaling through CD74 as well as T cell activation. These results suggest sex-dependent differences in which the absence of the MIF-1 and/or MIF-2 genotypes may permit stronger compensatory CD74-dependent EAE-inducing responses in males than in females. However, EAE severity in both sexes could still be reduced nearly to background (a "near cure") with DRα1-MOG-35-55 blockade of compensatory MIF and CD74-dependent factors known to attract peripheral inflammatory cells into the spinal cord tissue.

A selective small-molecule inhibitor of macrophage migration inhibitory factor-2 (MIF-2), a MIF cytokine superfamily member, inhibits MIF-2 biological activity.

Cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares pro-inflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. Here we describe the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC50 of 27 µm and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 µm) without influencing MIF-1-CD74 binding. Notably, 4-CPPC inhibited MIF-2-mediated activation of CD74 and reduced CD74-dependent signal transduction. These results open opportunities for development of more potent and pharmacologically auspicious MIF-2 inhibitors to investigate the distinct functions of this MIF family member in vivo.

Perturbation Theory/Machine Learning Model of ChEMBL Data for Dopamine Targets: Docking, Synthesis, and Assay of New l-Prolyl-l-leucyl-glycinamide Peptidomimetics.

Predicting drug-protein interactions (DPIs) for target proteins involved in dopamine pathways is a very important goal in medicinal chemistry. We can tackle this problem using Molecular Docking or Machine Learning (ML) models for one specific protein. Unfortunately, these models fail to account for large and complex big data sets of preclinical assays reported in public databases. This includes multiple conditions of assays, such as different experimental parameters, biological assays, target proteins, cell lines, organism of the target, or organism of assay. On the other hand, perturbation theory (PT) models allow us to predict the properties of a query compound or molecular system in experimental assays with multiple boundary conditions based on a previously known case of reference. In this work, we report the first PTML (PT + ML) study of a large ChEMBL data set of preclinical assays of compounds targeting dopamine pathway proteins. The best PTML model found predicts 50000 cases with accuracy of 70-91% in training and external validation series. We also compared the linear PTML model with alternative PTML models trained with multiple nonlinear methods (artificial neural network (ANN), Random Forest, Deep Learning, etc.). Some of the nonlinear methods outperform the linear model but at the cost of a notable increment of the complexity of the model. We illustrated the practical use of the new model with a proof-of-concept theoretical-experimental study. We reported for the first time the organic synthesis, chemical characterization, and pharmacological assay of a new series of l-prolyl-l-leucyl-glycinamide (PLG) peptidomimetic compounds. In addition, we performed a molecular docking study for some of these compounds with the software Vina AutoDock. The work ends with a PTML model predictive study of the outcomes of the new compounds in a large number of assays. Therefore, this study offers a new computational methodology for predicting the outcome for any compound in new assays. This PTML method focuses on the prediction with a simple linear model of multiple pharmacological parameters (IC50, EC50, Ki, etc.) for compounds in assays involving different cell lines used, organisms of the protein target, or organism of assay for proteins in the dopamine pathway.

From blood to brain through BBB and astrocytic signaling.

In this Festschrift, I discuss the career and guiding principles to which Abba J. Kastin has adhered during the last 20 years we worked together. I briefly describe the history of our joint laboratory group, the context of studies of peptide permeation across the blood-brain barrier (BBB), and newer developments in the BBB Group as Abba steps down after serving 35 years as the founding Editor-in-Chief for Peptides. Abba's BBB studies on peptides have contributed to concepts in the neuroendocrinology of feeding and developed information on molecular trafficking across BBB cells. The astroglial leptin signaling studies and the interactions of sleep and BBB are two major directions, whereas the long-term MIF-1 project demarcates a tortuous road on translational research.

An extraordinary relationship involving MIF-1 and other peptides.

In commemoration of Abba J. Kastin's exceptional service as the founding editor for the international journal Peptides, I review our collaborative work on how neuropeptides are involved in depression and other neuropsychiatric behavior. A special focus is on MIF-1 (prolyl-leucyl-glycinamide) that was discovered in the Kastin laboratory and shown effective to treat human depression with greater efficacy and faster onset of action than traditional antidepressants at the time of clinical trial. My personal reflection of the evolving changes of translational research on neuropeptides will hopefully provide some insight to young investigators.

Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH2 to the dopamine D2L receptor: evidence for the allosteric modulation of the dopamine receptor.

The present study was undertaken to investigate the mechanistic role of l-prolyl-l-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D(2L) receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride (1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [(125)I]-1b to an approximately 51kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D(2L)-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this approximately 51kDa protein to be the dopamine D(2L) receptor. Further evidence for binding of 1b to dopamine D(2L) was provided by samples immunoprecipitated with the D(2L) antibody. These samples were analyzed by western blotting in parallel with autoradiography of [(125)I]-1b labeled protein. Both methods revealed bands at approximately 51kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D(2L) receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity-labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D(2L) receptor.

Mass spectrometric quantification of MIF-1 in mouse brain by multiple reaction monitoring.

MIF-1 (Pro-Leu-Gly-NH(2)) has potent therapeutic effects in depression and Parkinson's disease, but its CNS sites of production are not yet clear. In this study, the concentration of MIF-1 in different brain regions was measured by the multiple reaction monitoring technique on a 4000 QTRAP mass spectrometer. The limit of quantification was 300 fg of MIF-1, and limit of detection was 60 fg. The low molecular weight fractions of tissue homogenates from different regions of mouse brain were analyzed. The concentration of MIF-1 ranged from 22+/-3 fg/microg protein in cerebral cortex to 930+/-60 fg/microg protein in the hypothalamus. Moderate concentrations were also detected in all other regions tested, including the striatum, thalamus, and hippocampus. By incubation of stable isotope-labeled oxytocin with tissue preparations, it was also confirmed that oxytocin at least partially contributed to the production of MIF-1 in the hypothalamus by action of peptidases. Regional differences were also found. The results are the first to show the ultrasensitive quantification of MIF-1 in different brain regions, and support the neuromodulatory actions of MIF-1 in the striatum.

Involvement of nitric oxide in the nociception of kyotorphin, TYR-CAV and MIF-s analogues in the rat spinal cord.

The spinal analgesic effects of Kyotorphin (Kyo) and Melanocyte-inhibiting factor (MIF-l) were studied during acute pain in rats chronically implanted with intrathecal (i.t.) cannulas. Kyo (5 micrograms), t-Cav (5 micrograms), Tyr-Cav (5 micrograms), L-NAME (1500 micrograms), MIF-Cav (200-400 micrograms) and MIF-sLeu (200 micrograms) exerted antinociceptive effects in both tests. The coadministration of Kyo + L-NAME enhanced the nociceptive effect compared with L-NAME (PP) or Kyo alone (PP, TF). The combination of Tyr-Cav + L-NAME enhanced the antinociceptive effect compared with L-NAME (PP) or Tyr-Cav alone (TF, PP). MIF-l (200 micrograms) had a weak antinociceptive effect in both tests. The coadministration of MIF-Cav + L-NAME enhanced the nociceptive effect compared with L-NAME (TF) or MIF-Cav alone (TF). The combination of MIF-sLeu + L-NAME enhanced the antinociceptive effect compared with L-NAME (TF) or MIF-sLeu alone (TF, PP). The results suggest that nitric oxide (NO) is involved in the antinociceptive effects of neuropeptides in the rat spinal cord.

Melanization stimulating factor (MSF) and melanization inhibiting factor (MIF) in the integument of fish.

The present studies were directed to demonstrate that adult fish skin contains putative factors that affect chromatophore and/or chromatoblast function. This hypothesis is based upon the possibility that hypo and/or hyperpigmented areas of the skin are so pigmented because of the localized expression of intrinsic factors that are either stimulatory or inhibitory to the differentiation of specific pigment cell types. In all the morphological and biochemical experiments carried out, we used culture media conditioned by dorsal (DCM) or ventral (VCM) skin from different species of fish. Both DCM and VCM were capable of stimulating differentiation of melanophores in neural crest explants. While the stimulation of melanization is an activity present in both dorsal and ventral skin, an inhibitory activity is also present in ventral skin at such a concentration that it overrides the stimulatory activity afforded by DCM. With biochemical assays, we demonstrated that the three important sequential enzymatic steps in melanogenesis are all stimulated by the conditioned media in a dose-dependent manner and this results in an increase in the amount of melanin present in cultured cells. The results of our investigations provide strong evidence that there are intrinsic pigment cell regulatory factors in the integument of fish, the inhibitory activity being stronger in the ventrum, and that those factors strongly influence, perhaps even determine, the pigment patterns of fish.

Inhibition of hematopoietic progenitor cell growth by Tyr-MIF, an endogenous opiate modulator, and its degradation products.

There is increasing evidence that neuronal factors can affect hematopoietic cell proliferation. Endogenous opioids with specificity for several opioid receptor classes were tested for their ability to inhibit murine and human hematopoietic progenitor cell proliferation. Tyr-MIF, an opioid tetrapeptide (H-Tyr-Pro-Leu-Gly-NH2), demonstrated a dose-dependent inhibition of colony formation at concentrations < 10 uM, inhibiting M-CSF and G-CSF-responsive progenitor cells equally. Tyr-MIF did not inhibit the number of colonies responsive to recombinant interleukin 3 (rmIL-3) or recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF), but significantly reduced colony size of GM-CSF responsive colonies. Colony formation by human low density and CD34+ marrow cells in response to G-CSF was also inhibited by Tyr-MIF and was more sensitive to inhibition than murine progenitor cells. Colony formation by single CD34+ cells was also inhibited by Tyr-MIF, indicating an effect directly on progenitor cells. Incubation of marrow cells in liquid culture and removal of Tyr-MIF prior to quantitating progenitor cell proliferation demonstrated that opioid-induced inhibition was reversible. The inhibitory effect of Tyr-MIF was not blocked by naloxone, a mu receptor specific antagonist, or diminished in mu opioid receptor deficient mice. HPLC analysis of cell-free culture medium containing Tyr-MIF showed no presence of the parent peptide after 24 h while progenitor cell inhibitory activity was retained. Analysis of potential degradation products of Tyr-MIF indicated that only H-Gly-NH9 or H-Gly-NH2 containing peptides inhibited colony forming unit (CFU) proliferation. These results indicate that Tyr-MIF is a reversible inhibitor of mature hematopoietic progenitor cell proliferation, and that this effect is most likely mediated by the degradation product H-Gly-NH2. Potential applications including protection of myeloid cells after cytosuppresive therapy are discussed.

Endomorphins, Met-enkephalin, Tyr-MIF-1, and the P-glycoprotein efflux system.

The P-glycoprotein (P-gp) transport system, responsible for the efflux of many therapeutic drugs out of the brain, recently has been shown to transport the endogenous brain opiate endorphin. We used P-gp knockout mice (Mdr1a) and their controls to determine where P-gp is involved in the saturable efflux systems of four other endogenous opiate-modulating peptides across the blood-brain barrier (BBB). After injection of endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)), endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)), Met-enkephalin (Tyr-Gly-Gly-Phe-Met-OH), and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH(2)) into the lateral ventricle of the mouse brain, residual radioactivity was measured at 0, 2, 5, 10, and 20 min later. The results showed no difference in the disappearance of any of these peptides from the brains of knockout mice compared with their controls. This demonstrates that unlike endorphin and morphine, P-gp does not seem to be required for the brain-to-blood transport of the endomorphins, Met-enkephalin, or Tyr-MIF-1 across the BBB.

Saturable brain-to-blood transport of endomorphins.

Opiate-modulating tetrapeptides such as tyrosine-melanocyte-stimulating hormone-release inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) are saturably transported from brain to blood. We examined whether two recently described endogenous opiate tetrapeptides with similar structures, the mu-specific endomorphins, also are transported across the blood-brain barrier (BBB). We found that the efflux rates of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were each self-inhibited by an excess of the respective endomorphin, thereby defining saturable transport. Cross-inhibition of the transport of each endomorphin by the other indicated shared transport. By contrast, no inhibition of the efflux of either endomorphin resulted from coadministration of Tyr-MIF-1, indicating that peptide transport system-1 (PTS-1) was not involved. Tyr-W-MIF-1, which is partially transported by PTS-1, significantly (P<0.01) decreased the transport of endomorphin-1 and tended (P=0.051) to decrease the transport of endomorphin-2, consistent with its role as both an opiate and antiopiate. Although involved in modulation of pain, coinjection of calcitonin gene-related peptide or constriction of the sciatic nerve did not appear to inhibit endomorphin efflux. Thus, the results demonstrate the existence of a new efflux system across the BBB which saturably transports endomorphins from brain to blood.

Regional differences in peptide degradation by rat cerebral microvessels: a potential novel regulatory mechanism for communication between blood and brain.

The blood-brain barrier (BBB), composed of the microvessels of cerebral capillary endothelial cells, regulates the passage of peptides into the brain in several ways, mainly by saturable transport or passive diffusion. Here we describe an additional mechanism by which this regulatory function can occur. Cerebral microvessels were isolated from different regions of the brain and incubated with the mu-opiate selective endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) or the opiate-modulating Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), both tetrapeptides selectively tritiated at the Pro. Degradation was determined by HPLC. For both peptides, the metabolism by microvessels from the cerebral cortex was much greater than that by microvessels from the hypothalamus or pons. For endomorphin-1, the least degradation was in the pons; for Tyr-MIF-1 there was no difference in metabolism by microvessels from the pons or hypothalamus. The results show a novel mechanism at the BBB by which the BBB can selectively regulate the activity of different peptides in different regions of the brain.

Withdrawal from alcohol in withdrawal seizure-prone and -resistant mice: evidence for enkephalin resistance.

Methionine enkephalin (Met-enkephalin) functions as an endogenous anticonvulsant. Peptide transport system-1 (PTS-1) is an important regulator of Met-enkephalin levels in brain and transports the peptide from brain to blood. In outbred mice, alcohol dependence is associated with decreased PTS-1 activity and increased levels of Met-enkephalin. In contrast, alcohol withdrawal is associated with recovery of PTS-1 activity, decreased levels of Met-enkephalin, and seizures. In this study, we examined the PTS-1/Met-enkephalin system in two replicates of withdrawal seizure-resistant (WSR) and withdrawal seizure-prone (WSP) mouse lines. We measured levels of preproenkephalin (PPE) mRNA and Met-enkephalin peptide in brain and the activity of PTS-1 during alcohol-naive, -dependent, and -withdrawal states. In alcohol-naive animals, Met-enkephalin levels were higher in WSP than in WSR mice. In alcohol-withdrawal animals, Met-enkephalin levels remained elevated in WSP mice, whereas they increased in WSR mice. Peptide levels were unrelated to levels of PPE mRNA or activity of PTS-1. Factorial analysis showed that proneness to seizures was genetically linked to Met-enkephalin levels in alcohol-naive, -dependent, and -withdrawing mice but not to mRNA levels or PTS-1 activity. Overall, these results may be explained by resistance to enkephalin in WSP mice and suggest that the dysregulation of the PTS-1/Met-enkephalin system contributes to susceptibility to seizures in WSP mice.

PLGamide characterization and role in osmoregulation in leech brain.

Neurons immunoreactive to an antiserum specifically directed against the Prolyl-Leucyl-Glycinamide peptide (PLGamide: Melanocyte Inhibiting Factor: MIF) were detected in the brain of the leech Theromyzon tessulatum. Radioimmunoassay titrations of the PLGamide-like material at different physiological stages of the life cycle indicated a maximal amount at stage 3B, which is correlated to phase of both maximal water uptake and coelomic vitellogenin accumulation. In vivo experiments demonstrate that this cerebral PLGamide-like material is an anti-diuretic factor that would act at stage 3B in order to permit a water uptake leading to water retention allowing coelomic yolk protein accumulation. In brains of the Gnatobdellid leech Hirudo medicinalis and the Pharyngobdellid leech Erpobdella octoculata, anti-PLGamide material was also detected with an amount not differing with the degree of sex maturation of the animals, confirming the link between osmoregulation and ovogenesis in rhynchobdellid leeches. Using a combination of biochemical techniques including high-pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts and Edman degradation, we demonstrated the presence of an authentic MIF-1 peptide in leech brain. Finally, since in vertebrates MIF-1 belongs to the non-classical opioid peptide family, we studied its binding displacement, in contrast to morphine, on mu-receptors and on nitric oxide (NO) release experiments in leech brain. PLGamide did not bind to mu-alkaloid opioid receptors and did not stimulate NO release.

Induction of Myc-intron-binding polypeptides MIBP1 and RFX1 during retinoic acid-mediated differentiation of haemopoietic cells.

Retinoic acid-mediated differentiation of HL60 cells is associated with an alteration of chromatin structure that maps to protein-binding sequences within intron I of the c-myc gene and with down-regulation of c-myc expression. By using HeLa cell extracts, we previously identified two polypeptides, designated MIBP1 (for Myc-intron-binding peptide) and RFX1, that interact in vivo and bind to the intron I element; we showed that tandem repeats of an MIBP1/RFX1-binding site can exhibit silencer activity on a heterologous promoter. Here we demonstrate that p160 MIBP1 and p130 RFX1 are absent from undifferentiated HL60 cells. In addition, we show that treatment with retinoic acid induces both MIBP1 and RFX1 protein, as well as their DNA-binding activity, upon granulocytic differentiation of HL60 cells, with a gel mobility pattern identical to that of HeLa cells. In the absence of p160 MIBP1 and p130 RFX1, we observed that the altered gel mobility-shift pattern detected in undifferentiated HL60 cells reflects the binding of two novel polypeptides, p30 and p97, that can be cross-linked to the same recognition intron sequence. We also show that the time course of MIBP1 and RFX1 induction is inversely correlated with the down-regulation of c-myc levels during the treatment of HL60 cells with retinoic acid.

Peptide transport system-1 (PTS-1) for Tyr-MIF-1 and Met-enkephalin differs from the receptors for either.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Met-enkephalin share a saturable transport system (peptide transport system-1, PTS-1) across the blood-brain barrier but do not readily bind to each other's receptors. This information allows the unique opportunity to differentiate the transport protein(s) from the receptors for either peptide in brain endothelial cells. PTS-1 was studied in vitro by allowing radiolabeled Tyr-MIF-1 (125I-Tyr-MIF-1) to bind to the solubilized proteins of isolated murine brain microvessels in the presence or absence of potential inhibitors. Sephadex chromatography separated bound from free labeled peptide. The binding was saturable as shown by inhibition with increasing concentrations of unlabeled Tyr-MIF-1. 125I-Tyr-MIF-1 binding was not inhibited by an unrelated peptide or iodo-tyrosine. D-Tyr-MIF-1 had no effect, demonstrating the stereospecificity of the system. Met-enkephalin decreased the binding of 125I-Tyr-MIF-1 to 84.4% of total, whereas Leu-enkephalin was without effect. Agonists for the mu, delta, and kappa opiate receptors did not change the binding, indicating that the proteins which bound to 125I-Tyr-MIF-1 were not endogenous opiate receptors. The results indicate that, in vitro, Tyr-MIF-1 binds to brain microvessel proteins with characteristics similar to PTS-1.

Modulation of agonist stimulated adenylyl cyclase and GTPase activity by L-pro-L-leu-glycinamide and its peptidomimetic analogue in rat striatal membranes.

L-prolyl-L-leucyl-glycinamide (PLG), also known as melanocyte-stimulating hormone release inhibiting factor (MIF-1), is an endogenous brain tripeptide. Previous studies have shown that PLG, and its peptidomimetic analogues, render dopamine D2 receptors more responsive to agonists by maintaining the high-affinity binding state of the receptors. In the present study, we examined the effect PLG and its analogue 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetam ide (PAOPA) on dopamine-stimulated adenylyl cyclase and NPA-stimulated GTPase activity in rat striatal membranes. Dopamine-stimulated adenylyl cyclase activity was inhibited by both PLG and PAOPA in a dose-dependent manner, whereas R(-)-propylnorapomorphine (NPA)-stimulated low Km GTPase activity was significantly increased by 1 microM PLG or 1 nM PAOPA. These results suggest that PLG and PAOPA maintain the high affinity state of the D2 receptor by increasing GTP hydrolysis through stimulation of agonist-induced GTPase activity.

Regional differences in the metabolism of Tyr-MIF-1 and Tyr-W-MIF-1 by rat brain mitochondria.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) are endogenous neuropeptides with opiate modulating and other CNS effects. After incubation of the tritiated tetrapeptides with fractions of tissue from different areas of rat brain, formation of the metabolites was determined by HPLC. Marked regional differences in degradation were found for both peptides. The metabolism of Tyr-MIF-1, resulting in the formation of the biologically active MIF-1 (Pro-Leu-Gly-NH2), was greater in the mitochondrial than in the synaptosomal fractions. In the mitochondrial fraction, about twice as much MIF-1 was formed in brain cortex than in striatum, diencephalon, or midbrain/pons medulla. These results, showing differential metabolism in various areas of the brain, indicate another means for regulation of the concentrations of neuropeptides.