Delta 9-tetrahydrocannabinol-induced MAPK/ERK and Elk-1 activation in vivo depends on dopaminergic transmission.

It is now well established that central effects of Delta 9-tetrahydrocannabinol (THC), the main psychoactive component of marijuana, are mediated by CB1 cannabinoid receptors. However, intraneuronal signalling pathways activated in vivo by THC remain poorly understood. We show that acute administration of THC induces a progressive and transient activation (i.e. phosphorylation) of the mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) in the dorsal striatum and the nucleus accumbens (NA). This activation, corresponding to both neuronal cell bodies and the surrounding neuropil, is totally inhibited by the selective antagonist of CB1 cannabinoid receptors, SR 141716A. However, blockade of dopaminergic (DA) D1 receptors by administration of SCH 23390, prior to THC, totally prevents ERK activation in the striatum, thus demonstrating a critical involvement of DA systems in THC-induced ERK activation. DA-D2 and glutamate receptors of NMDA subtypes also participate, albeit to a lesser extent, to THC-induced ERK activation in the striatum, as shown after injection of selective antagonists (raclopride and MK801, respectively). Furthermore, THC-induced phosphorylation of the transcription factor Elk-1, and up-regulation of zif268 mRNA expression are blocked by SL327, a specific inhibitor of MAPK/ERK kinase (MEK), the upstream kinase of ERK, as well as SCH 23390. Finally, using the place-preference paradigm, we show that ERK inhibition blocks THC-induced rewarding properties. Altogether, our data strongly support that ERK activation in the striatum is critically involved in long-term neuronal adaptive responses underlying THC-induced long-term behaviours.

Lhx9 and Lhx9alpha LIM-homeodomain factors: genomic structure, expression patterns, chromosomal localization, and phylogenetic analysis.

Lhx9 is a LIM-homeodomain (LIM-hd) transcription factor expressed in the embryonic mouse brain. We report the isolation of Lhx9alpha, a cDNA encoding a truncated isoform of Lhx9 that lacks the recognition helix of the homeodomain and differs from Lhx9 cDNA in its 3'-coding and 3'-UTR sequences. Isolation of the Lhx9 gene showed that Lhx9 and Lhx9alpha are coded by six exons spanning 10 kb of genomic sequence and that Lhx9alpha is an isoform generated by alternative splicing of the fifth exon. Lhx9 was mapped to the subtelomeric region of chromosome 1. Further molecular analysis showed that Lhx9 is a new candidate gene for the unidentified dreher (dr) mutation in mouse. The comparison of genomic structure and molecular phylogenetic analysis led to the identification of six groups of LIM-hd proteins, a basis for further classification and knowledge of their evolutionary relationships. To investigate a possible role for Lhx9alpha, the expression patterns of Lhx9 and Lhx9alpha were compared during embryogenesis. Lhx9alpha was expressed at lower levels than Lhx9, with a similar but distinct pattern in the brain, especially in the neocortex. We suggest that Lhx9alpha could function as an endogenous dominant-negative form of Lhx9 during development, both to regulate in space and time the transcriptional effects of Lhx9 and to add a degree of refinement to the LIM-hd code.

Lhx9: a novel LIM-homeodomain gene expressed in the developing forebrain.

A novel LIM-homeodomain gene, Lhx9, was isolated by degenerate RT-PCR followed by mouse embryonic library screening. Lhx9 cDNA encodes a protein that is most closely related to Drosophila apterous and rodent Lhx2 proteins. The Lhx9 spatiotemporal pattern of expression during embryogenesis was similar but distinct from Lhx2. Highest expression levels were found in the diencephalon, telencephalic vesicles, and dorsal mesencephalon. Domains of expression respected the proposed neuromeric boundaries (). Lhx9 was also expressed in the spinal cord, forelimb and hindlimb mesenchyme, and urogenital system. Although Lhx9 expression was sustained in diencephalon and mesencephalon from embryonic day 10.5 (E10.5) to postnatal stages, it was transient in the future cerebral cortex, where it was turned off between E14.5 and E16.5. Lhx9 expression was highest if not exclusively located (depending on the region of interest) in the intermediate and mantle zones, as opposed to the mitotic ventricular zone. Lhx9 protein was tested for interaction with the recently discovered cofactors of LIM-homeodomain proteins and was found to interact strongly both with CLIM1 and CLIM2. The expression pattern and structural characteristics of Lhx9 suggest that it encodes a transcription factor that might be involved in the control of cell differentiation of several neural cell types. Furthermore, Lhx9 protein could act in a combinatorial manner with other LIM-homeodomain factors expressed in overlapping pattern.

Extracellular signal-regulated kinase (ERK) controls immediate early gene induction on corticostriatal stimulation.

Activity-dependent changes in neuronal structure and synaptic remodeling depend critically on gene regulation. In an attempt to understand how glutamate receptor stimulation at the membrane leads to gene regulation in the nucleus, we traced intracellular signaling pathways targeting DNA regulatory elements of immediate early genes (IEGs). For this purpose we used an in vivo electrical stimulation of the glutamatergic corticostriatal pathway. We show that a transient activation of extracellular signal-regulated kinase (ERK) proteins (detected by immunocytochemistry with an anti-active antibody) is spatially coincident with the onset of IEG induction [c-fos, zif 268, and map kinase phosphatase-1 (MKP-1) detected by in situ hybridization] in the striatum, bilaterally. Both Elk-1 and CREB transcription factors (targeting SRE and CRE DNA regulatory elements, respectively) were hyperphosphorylated in register with ERK activation and IEG mRNA induction. However, their hyperphosphorylation occurred in different subcellular compartments: the cytoplasm and the nucleus for Elk-1 and the nucleus for CREB. The role of the ERK signaling cascade in gene regulation was confirmed after intrastriatal and unilateral injection of the specific ERK inhibitor PD 98059, which completely abolished c-fos, zif 268, and MKP-1 mRNA induction in the injected side. Of interest, both Elk-1 and CREB hyperphosphorylation also was impaired after PD 98059 injection. Thus two different ERK modules, one depending on the cytoplasmic activation of Elk-1 and the other one depending on the nuclear activation of CREB, control IEG transcriptional regulation in our model. Our findings provide significant insights into intracellular mechanisms underlying synaptic plasticity in the striatum.

In vivo expression and regulation of Elk-1, a target of the extracellular-regulated kinase signaling pathway, in the adult rat brain.

The transcription factor Elk-1, a nuclear target of extracellular-regulated kinases (ERKs), plays a pivotal role in immediate early gene induction by external stimuli. Notably, the degree of phosphorylation of Elk-1 is tightly correlated with the level of activation of transcription of c-fos by proliferative signals. No data yet indicate the role of Elk-1 in the adult brain in vivo. To address this question, we have analyzed in the present work (1) Elk-1 mRNA and protein expression in the adult rat brain, and (2) the regulation of Elk-1 (i.e., its phosphorylation state) in an in vivo model of immediate early gene (IEG) induction: an electrical stimulation of the cerebral cortex leading to c-fos and zif268 mRNA induction in the striatum. Using in situ hybridization, we show that Elk-1 mRNA is expressed in various brain structures of adult rat, and that this expression is exclusively neuronal. We demonstrate by immunocytochemistry using various specific Elk-1 antisera that the protein is not only nuclear (as shown previously in transiently transfected cell lines) but is also present in soma, dendrites, and axon terminals. On electrical stimulation of the glutamatergic corticostriatal pathway, we show a strict spatiotemporal correspondence among ERK activation, Elk-1 phosphorylation, and IEG mRNA induction. Furthermore, both activated proteins, analyzed by immunocytochemistry, are found in cytosolic and nuclear comparments of neuronal cells in the activated area. Our data suggest that the ERK signaling pathway plays an important role in regulating genes controlled by serum response element sites via phosphorylation of Elk-1 in vivo.

Effect of electrical stimulation of the cerebral cortex on the expression of the Fos protein in the basal ganglia.

The protein Fos is a transcription factor which is quickly induced in response to a variety of extracellular signals. Since this protein is expressed in a variety of neuronal systems in response to activation of synaptic afferents, it has been suggested that it might contribute to activity-dependent plasticity in neural networks. The present study investigated the effect of cortical electrical stimulation on the expression of Fos in the basal ganglia in the rat, a group of structures that participate in sensorimotor learning. Results show that the repetitive application of electrical shocks in restricted areas of the cerebral cortex induces an expression of Fos mostly confined to the striatum and the subthalamic nucleus. The induction which can be elicited from different cortical areas (sensorimotor, auditory and limbic areas) does not require particular temporal patterns of stimulation but rather depends on the total number of shocks delivered during a given period of time. Moreover, it appears to be rather independent of the number of spikes discharged by the activated cells. In the striatum, the distribution of immunoreactive neurons is precisely delineated and conforms to the known topographical organization of stimulated corticostriatal projections. As demonstrated using a variety of double labelling techniques (combination of the immunocytochemical detection of Fos with the autoradiography of mu opioid receptors, calbindin immunocytochemistry, in situ hybridization of preproenkephalin and preprotachykinin A messenger RNAs), striatal neurons which express Fos are mostly localized in the matrix compartment and concern equally enkephaline and substance P containing efferent neurons. In the subthalamic nucleus, Fos expression evoked by cortical stimulation is also confined to discrete regions of the nucleus, the localizations corresponding to the primary projection site of the stimulated cortical cells. These results indicate that in addition to its phasic synaptic influence on the basal ganglia, the cerebral cortex could exert a long-term effect on the functional state of this system via a genomic control. Since the basal ganglia are involved in sensorimotor learning and motor habit formation, it is tempting to speculate that the activity-dependent Fos induction at corticostriatal and subthalamic synapses may contribute to consolidate the functionality of the neuronal networks activated during the completion of given motor tasks.

Molecular analysis of the multiple Golf alpha subunit mRNAs in the rat brain.

The alpha subunit of GTP-binding protein Golf (G alpha olf), identified in the olfactory epithelium, in which it is supposed to mediate odorant-generated adenylyl cyclase activations, is much more expressed in the striatum than G alpha s, the classical stimulatory G protein alpha subunit (Hervé et al., J Neurosci., 13 (1993) 2237-2248). Four species of G alpha olf messenger RNA (mRNA) were observed by Northern blot in the rat striatum. Analysis of striatal G alpha olf cDNA clones demonstrated that despite extensive variations in their 5' and 3' untranslated regions, these four G alpha olf mRNAs encode the same G alpha olf polypeptide. One of the four mRNA species, which was selectively observed in the striatum, is generated by a novel promoter whose activity was barely detectable in the olfactory epithelium. Surprisingly, this promoter generates an unexpectedly high proportion of transcripts in which the first intron is unspliced. The retention of intron was found to abolish the translation of G alpha olf mRNA in the reticulocyte lysate system, suggesting that it could be involved in the down regulation of G alpha olf expression in the striatum. Furthermore, a new polyadenylation site with a non canonical sequence, AATACA, was found to be responsible of the two shortest forms of G alpha olf mRNA. In conclusion, we have shown that the G alpha olf proteins present in the striatum and the olfactory epithelium are identical and that multiple variations in the untranslated parts of the mRNAs could affect G alpha olf expression.

Chronic blockade of muscarinic cholinergic receptors by systemic trihexyphenidyl (Artane) administration modulates but does not mediate the dopaminergic regulation of striatal prepropeptide messenger RNA expression.

A striatal dopaminergic denervation leads to changes in the expression of messenger RNA encoding prepropeptides contained in striatal efferent neurons. Such a dopaminergic lesion also abolishes a functional equilibrium between dopaminergic and cholinergic transmissions, generally believed to operate within the neostriatum, which constitutes the theoretical basis for the clinical use of antimuscarinic drugs in extrapyramidal diseases. It is possible, therefore, that changes in prepropeptide messenger RNA expression are mediated by an alteration in cholinergic transmission. To test this hypothesis, we have examined in rats whether trihexyphenidyl, an antimuscarinic drug of wide clinical use, can counteract the changes in preproenkephalin, preprotachykinin and preprodynorphin messenger RNA expression produced by a unilateral 6-hydroxydopamine lesion of substantia nigra dopaminergic neurons. Two weeks after the lesion, trihexyphenidyl was continuously administered through an osmotic minipump (5 mg/day for 15 days) to half of the lesioned and sham-operated rats, the other half receiving the vehicle. Using quantitative in situ hybridization histochemistry, messenger RNAs were analysed at two rostrocaudal levels (anterior and central) of the neostriatum. In parallel, M1 muscarinic receptors were measured by autoradiography of [3H]pirenzepine binding sites. In sham-operated rats, trihexyphenidyl administration produced a significant increase (17-27%) in M1 binding sites. In addition, preproenkephalin messenger RNA levels were decreased (-38%) in the central part, while preprodynorphin messenger RNA levels were significantly increased (+22%) at both striatal levels. In 6-hydroxydopamine-lesioned rats, the expected changes in messenger RNAs were observed when ipsi- versus contralateral side values were compared, but changes were not always detected when comparison was established between values from the dopamine-denervated neostriatum and those from sham-operated rats. The trihexyphenidyl administration in 6-hydroxydopamine-lesioned animals was unable to reproduce the up-regulation of M1 receptors, even in the intact neostriatum. This antimuscarinic treatment further increased preproenkephalin messenger RNA levels in the denervated anterior neostriatum, amplifying the ipsi- versus contralateral difference. It also potentiated the imbalance in preprotachykinin messenger RNA expression, mainly as a result of an increase of preprotachykinin messenger RNA levels in the intact neostriatum. In contrast, trihexyphenidyl treatment by increasing preprodynorphin messenger RNA in both neostriata abolished the ipsi- versus contralateral difference observed in lesioned rats. In conclusion, with the exception of preprodynorphin messenger RNA, trihexyphenidyl treatment was unable to counteract the imbalance in prepropeptide messenger RNA expression produced by a unilateral striatal dopaminergic denervation and even amplified this effect. These results question the neostriatum as the site of action of antimuscarinic drugs in producing their therapeutic effect in extrapyramidal syndromes.

The rat nucleus accumbens: two levels of complexity in the distribution of glutamic acid decarboxylase (67 kDa) and preproenkephalin messenger RNA.

The distribution of messenger RNAs (mRNAs) encoding the 67 kDa isoform of glutamic acid decarboxylase (GAD67), a rate-limiting enzyme in the biosynthesis of gamma-aminobutyric acid, and preproenkephalin (PPE), the precursor of enkephalin, was analysed in the rat nucleus accumbens (NAS) taking in account its compartmentation in 'core' (NAS(core)) and 'shell' (NASshell). While GAD67 mRNA levels were more abundant (+48%) in NASshell than in NAS(core), hybridization signals for PPE mRNA were mainly found in NAScore. At a cellular level, hybridization signals for GAD67 mRNA were present in 75% and 80% of neurons NASshell and NAS(core), respectively. The level of GAD67 mRNA found higher in the NASshell corresponds in fact to a greater density of neurons in NASshell than in the NAS(core). The hybridization signals for PPE mRNA were observed in very few neurons of NASshell and in around half of the neurons in NAS(core). Besides this medio to lateral difference, GAD67 and PPE mRNAs were distributed in NAS(core) according to a rostro-caudal gradient, the content of both mRNAs being higher in the rostral part than the caudal part of this area.

GABA interneurons in the rat medial frontal cortex: characterization by quantitative in situ hybridization of the glutamic acid decarboxylase (GAD67) mRNA.

In situ hybridization of mRNA encoding one isoform of glutamic acid decarboxylase (GAD67) was performed in the rat medial frontal cortex (MFC) to characterize GABA interneurons. Qualitatively, the labelling obtained with a [35S]cDNA probe was in register with neurons and was never associated with glial cells. No obvious differences in the density of labelled cells were observed between the different areas of the MFC examined (infralimbic, prelimbic, anterior cingulate and precentral medial) and between the various cortical layers. Grain counting was performed on single cells in the various layers of the prelimbic and the anterior cingulate area, two main areas of the MFC. According to their grain density, neurons were arbitrarily classified as low, high and very high GAD67 mRNA content. The neurons with the high GAD67 mRNA content corresponded to around 50% of the labelled cells in all the layers and in both areas. In the prelimbic area, the neuronal population with a low GAD67 mRNA content varied from 50% in layers I and II-III to 40% in layers V-VI whereas the very high GAD67 mRNA content neurons corresponded to around 5% of the labelled neurons in all layers. In the anterior cingulate area the neuronal population showing low GAD67 mRNA content varied from 35% in layers I and II-III to 20% in layers V-VI. In this area, neurons with a very high GAD67 mRNA content were more numerous than in the prelimbic area: they varied from 15% in layers I and II-III to 30% in layers V-VI. Parallel to the presence of very highly labelled cells, GAD enzymatic activity measured both in the presence and in the absence of pyridoxal 5'-phosphate was higher in the anterior cingulate area than in the prelimbic area. The heterogeneity of GAD67 mRNA content at the cellular level might underlie the existence of subpopulations of GABA interneurons in the MFC and suggests a higher GABAergic inhibitory control in the anterior cingulate area than in the prelimbic area.

Haloperidol increases PPE mRNA levels in the caudal part of the nucleus accumbens in the rat.

Dopamine exerts a tonic inhibitory control in the regulation of mRNA encoding preproenkephalin (PPE), the precursor of enkephalin, in the dorsal part of the rat striatum. Less is known about the role of this amine in the regulation of PPE mRNA in the ventral part of the striatum which corresponds to the nucleus accumbens. In this study, the effects of an interruption of dopamine transmission by haloperidol (a dopaminergic receptor antagonist) were examined in the rat nucleus accumbens. The levels of PPE mRNA were analysed by in situ hybridization histochemistry by taking into account the complexity of the nucleus accumbens, i.e. by differentiating the rostro-caudal extension of the structure. It was found that PPE mRNA content was increased significantly only in the caudal part of the nucleus, indicating rostro-caudal differences in the haloperidol effects. The differential reactivity of PPE mRNA to dopaminergic receptor blockade and to a lesion of dopaminergic neurones along the rostrocaudal axis of the ventral striatum is discussed.

Parallel decrease of glutamic acid decarboxylase and preproenkephalin mRNA in the rat striatum following chronic treatment with a dopaminergic D1 antagonist and D2 agonist.

The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long-term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (-30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (-38 and -57%, respectively) as well as in the ventral (-70 and -60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (-41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.

Comparative development of D1-dopamine and mu opiate receptors in normal and in 6-hydroxydopamine-lesioned neonatal rat striatum: dopaminergic fibers regulate mu but not D1 receptor distribution.

The postnatal development of D1 dopaminergic receptors (D1 receptors) was investigated in the rat striatum in relation to distribution of mu opiate receptor patches and islandic tyrosine hydroxylase (TH)-immunoreactive fibers. The possible influence of dopaminergic (DA) fibers originating from the substantia nigra on the postnatal distribution of striatal D1 and mu receptors was also examined by producing an early 6-hydroxydopamine (6-OHDA) lesion of DA fibers. D1 and mu receptors were labeled with selective ligands: [3H]SCH 23390 and [3H]DAGO, respectively. During the first postnatal week, control rats showed patches of dense D1 binding sites in the entire rostro-caudal extension of the striatum. The localization of D1 receptor patches corresponded to striosomes identified by TH-immunoreactive islands. The striatal distribution of mu receptors was relatively homogeneous at postnatal day 0 (P0) but was clearly patchy at P3-P4. During the second postnatal week the striosomal pattern of D1 binding sites disappeared along a dorso-ventral gradient whereas mu binding sites remained distributed in patches. Densitometric measurements showed that there was a parallel increase of D1 binding sites in both striosomes and the surrounding matrix from P0 to P4. The disappearance of D1 receptor patches observed in the dorsal striatum at P9 was due to a faster increase of D1 binding sites in the matrix than in striosomes between P4 and P9 whereas a significant difference was still observed between these two compartments in the ventral striatum of P9 rats. During the third postnatal week, the density of D1 binding sites still increased but became progressively uniform in the whole striatum. The intrastriatal injection of 6-OHDA in 2-day-old rats produced a local disappearance of TH-immunoreactive fibers in the striatum and a distal degeneration of TH-immunoreactive cell bodies in the substantia nigra. However an early lesion of striatal DA fibers did not modify the pattern of development or the density of D1 binding sites during the postnatal period examined (1 and 3 weeks after the lesion). The distribution of mu receptors was unchanged 1 week after the lesion but showed a clear disorganization 3 weeks after the lesion. We discuss the differential influence of DA fibers on the distribution of D1 and mu receptors in the rat striatum and the possible role of DA in the regulation of the expression of mu receptors.

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex.

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate cyclase of bovine adrenal cortex plasma membranes. Divergence between corticotropin and fluoride combined effects with forskolin.

The diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9-fold with a concentration producing half-maximum effect (ED50) of about 4 microM. The effects of forskolin and the fully active corticotropin fragment ACTH (I 24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms.

Identification of haptoglobin in chicken serum and specificity of the chicken haptoglobin-hemoglobin complex formation.

1. The presence of haptoglobin in chicken serum has been demonstrated by three different techniques: gel filtration, cellulose acetate electrophoresis and fluorescence quenching. 2. Chicken haptoglobin shows a narrow species specificity; it binds only avian and reptilian but not mammalian hemoglobins. 3. Haptoglobin seems to have been subjected to profound changes during the course of evolution.

Studies on hemoglobin tryptophanyl contact residues in the haptoglobin-hemoglobin complex.

Hemoglobin and apohemoglobin bind heptoglobin in the same molar ratio. Structural studies on haptoglobin-hemoglobin complex do not suggest any important structural changes in either protein upon binding. However, when apohemoglobin is bound to haptoglobin, a marked reduction in secondary structure, attributed to unfolding of globin chains, has been observed. Here we describe some properties of the haptoglobin-apohemoglobin (Hp-apoHb) complex, prepared by isoelectric focusing in the presence of an excess of haptoglobin. This complex does not exhibit the irreversibility of complexes obtained with hemoglobin in identical experimental conditions. The 'freezing' of the conformation of apohemoglobin upon binding to haptoglobin has been studied by fluorescence quenching experiments carried out in the presence of 8 M acrylamide. Changes in conformation of haptoglobin upon binding to apohemoglobin have been detected by titration of the exposed tryptophans using N-bromosuccinimide. Comparison of the additivity of exposed tryptophans in the complexes reveal that two tryptophans become inaccessible in the complex formation of haptoglobin with hemoglobin but not with apohemoglobin. These tryptophans, probably located on the alpha1beta2 contact interface of hemoglobin, have been tentatively identified as Trp-C3(37)beta.