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PubMed2006

The centrifugal visual system of vertebrates: a comparative analysis of its functional anatomical organization.

The present review is a detailed survey of our present knowledge of the centrifugal visual system (CVS) of vertebrates. Over the last 20 years, the use of experimental hodological and immunocytochemical techniques has led to a considerable augmentation of this knowledge. Contrary to long-held belief, the CVS is not a unique property of birds but a constant component of the central nervous system which appears to exist in all vertebrate groups. However, it does not form a single homogeneous entity but shows a high degree of variation from one group to the next. Thus, depending on the group in question, the somata of retinopetal neurons can be located in the septo-preoptic terminal nerve complex, the ventral or dorsal thalamus, the pretectum, the optic tectum, the mesencephalic tegmentum, the dorsal isthmus, the raphé, or other rhombencephalic areas. The centrifugal visual fibers are unmyelinated or myelinated, and their number varies by a factor of 1000 (10 or fewer in man, 10,000 or more in the chicken). They generally form divergent terminals in the retina and rarely convergent ones. Their retinal targets also vary, being primarily amacrine cells with various morphological and neurochemical properties, occasionally interplexiform cells and displaced retinal ganglion cells, and more rarely orthotopic ganglion cells and bipolar cells. The neurochemical signature of the centrifugal visual neurons also varies both between and within groups: thus, several neuroactive substances used by these neurons have been identified; GABA, glutamate, aspartate, acetylcholine, serotonin, dopamine, histamine, nitric oxide, GnRH, FMRF-amide-like peptides, Substance P, NPY and met-enkephalin. In some cases, the retinopetal neurons form part of a feedback loop, relaying information from a primary visual center back to the retina, while in other, cases they do not. The evolutionary significance of this variation remains to be elucidated, and, while many attempts have been made to explain the functional role of the CVS, opinions vary as to the manner in which retinal activity is modified by this system.

Animals↗

PubMed2006

The evolution of the centrifugal visual system of vertebrates. A cladistic analysis and new hypotheses.

In a recent review of the available data concerning the centrifugal visual system (CVS) of vertebrates [Repérant, J., Ward, R., Miceli, D., Rio, J.P., Médina, M., Kenigfest, N.B., Vesselkin, N.P., 2006. The centrifugal visual system of vertebrates: a comparative analysis of its functional anatomical organization, Brain Res. Rev. 52, 1-57], we have shown that this feature of the visual system is not a particularity of birds, but is a permanent component of the vertebrate central nervous system which nevertheless shows considerable morphological and functional variation from one taxonomic group to another. Given these findings, the primary objective of the present article is an attempt to specify the evolutionary significance of this phylogenetic diversity. We begin by drawing up an inventory of this variation under several headings: the intracerebral location of the retinopetal neurons; the mode of intra-retinal arborizations of the centrifugal fibres and the nature of their targets; their neurochemical properties; and the afferent supplies of these neurons. We subsequently discuss these variations, particularly that of the intracerebral location of the retinopetal neurons during development and in adult forms, using the neuromeric terminology and in the framework of cladistic analysis, and seek to interpret them in a phylogenetic context. From this analysis, it becomes evident that the CVS is not a homogeneous entity formed by neurons with a common embryological origin, but rather a collection of at least eight distinct subsystems arising in very different regions of the neuraxis. These are the olfacto-retinal, dorsal thalamo-retinal, ventral thalamo-retinal, pretecto-retinal, tecto-retinal, tegmento-mesencephalo-retinal, dorsal isthmo-retinal and ventral isthmo-retinal systems. The olfacto-retinal system, which is probably absent in Agnatha, appears to be a pleisiomorphic characteristic of all Gnathostomata, while on the other hand the tegmento-mesencephalo-retinal system appears to be present only in Agnatha. Our cladistic analysis also shows that the remaining six subsystems are polyphyletic in origin and have arisen independently on several occasions in different radiations of Gnathostoma. In conclusion, we suggest that, in the course of the palaeontological history of vertebrates, these different retinopetal pathways have been selected on the basis of widely different environmental pressures which remain to be identified.

Animals↗

PubMed2005

Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb.

The centrifugal systems innervating the olfactory bulb are important elements in the functional regulation of the olfactory pathway. In this study, the selective innervation of specific glomeruli by serotonergic, noradrenergic and cholinergic centrifugal axons was analyzed. Thus, the morphology, distribution and density of positive axons were studied in the glomerular layer of the main olfactory bulb of the rat, using serotonin-, serotonin transporter- and dopamine-beta-hydroxylase-immunohistochemistry and acetylcholinesterase histochemistry in serial sections. Serotonin-, serotonin transporter-immunostaining and acetylcholinesterase-staining revealed a higher heterogeneity in the glomerular layer of the main olfactory bulb than previously reported. In this sense, four types of glomeruli could be identified according to their serotonergic innervation. The main distinctive feature of these four types of glomeruli was their serotonergic fibre density, although they also differed in their size, morphology and relative position throughout the rostro-caudal main olfactory bulb. In this sense, some specific regions of the glomerular layer were occupied by glomeruli with a particular morphology and a characteristic serotonergic innervation pattern that was consistent from animal to animal. Regarding the cholinergic system, we offer a new subclassification of glomeruli based on the distribution of cholinergic fibres in the glomerular structure. Finally, the serotonergic and cholinergic innervation patterns were compared in the glomerular layer. Sexual differences concerning the density of serotonergic fibres were observed in the atypical glomeruli (characterized by their strong cholinergic innervation). The present report provides new data on the heterogeneity of the centrifugal innervation of the glomerular layer that constitutes the morphological substrate supporting the existence of differential modulatory levels among the entire glomerular population.

Acetylcholine↗

PubMed2000

Bridge to recovery for postcardiotomy failure: is there still a role for centrifugal pumps?

BACKGROUND: Early implantation of centrifugal devices in patients with postcardiotomy cardiogenic shock may provide a bridge to recovery and allow subsequent long-term survival. METHODS: Since January 1989, 62 patients were supported with centrifugal pumps because of failure to wean from cardiopulmonary bypass. Indications were postcardiotomy cardiogenic shock (PCCS) (n = 60), bridge to cardiac retransplantation (n = 1), and right ventricular failure (n = 1). Patients' ages ranged from 23 to 78 years; 40 were men (65%), and 22 were women (35%). Twenty-two patients (35%) had a left ventricular assist device; 9 patients (15%) had a right ventricular assist device; and 31 patients (50%) had a biventricular assist device. Length of support ranged from 1 day to 19 days. RESULTS: Forty-two patients (68%) were weaned successfully; 27 patients survived to discharge (44%). Complications included bleeding (n = 41, 66%), renal failure (n = 28, 45%), and respiratory failure (n = 26, 42%). Currently, 23 patients survived 10 or more years (n = 1), 6 to 10 years (n = 7), 1 to 5 years (n = 10), and less than 1 year (n = 5). CONCLUSIONS: Centrifugal pumps are available, easy to use, and relatively inexpensive. Our experience justifies their continued use as a bridge to recovery for patients with postcardiotomy cardiogenic shock, despite the availability and increasing use of more expensive devices.

Adult↗

PubMed1998

Serum S100beta release after coronary artery bypass grafting: roller versus centrifugal pump.

BACKGROUND: Microemboli generated during cardiopulmonary bypass (CPB) are implicated in the cerebral injury seen after coronary artery bypass grafting. Centrifugal pumps generate fewer microemboli than roller pumps. Increased S100beta levels have been reported after coronary artery bypass grafting, with levels greater than 1 ng/mL resulting in poorer neuropsychologic outcome. This study investigated the potential neurologic benefits of centrifugal pumps, by using S100beta as a marker for cerebral injury. METHODS: Thirty-two patients who had coronary artery bypass grafting were randomly assigned to two groups. Serial blood samples (preoperative, end of bypass, 30 minutes, and 2 and 24 hours after cardiopulmonary bypass) were taken and the serum analyzed for S100beta using a new immunoluminometric assay. RESULTS: Both groups were matched for age, number of grafts, and cardiopulmonary bypass and cross-clamp times. Postoperative serum S100beta levels were significantly higher in both groups than preoperative levels. Peak S100beta levels did not correlate with cardiopulmonary bypass time; however, 24-hour S100beta levels correlated with intubation time r = 0.40, p = 0.04). Th ere was no significant difference in S100beta levels between the groups at any of the time points. CONCLUSIONS: S100beta levels increased after coronary artery bypass grafting. Centrifugal pumps do not significantly decrease S100beta release. Persistently increased S100beta levels are associated with longer intubation times.

Calcium-Binding Proteins↗

PubMed2000

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation.

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.

Animals↗

PubMed1997

Distribution of centrifugal neurons targeting the soma clusters of the olfactory midbrain among decapod crustaceans.

To determine the distribution of two systems of centrifugal neurons innervating the soma clusters of the olfactory midbrain across decapod crustaceans, brains of the following nine species comprising most infraorders were immunostained with antibodies against dopamine and the neuropeptides substance P and FMRFamide: Macrobrachium rosenbergii, Homarus americanus, Cherax destructor, Orconectes limosus, Procambarus clarkii, Astacus leptodactylus, Carcinus maenas, Eriocheir sinensis and Pagurus bernhardus. One system consisting of several neurons with dopamine-like immunoreactivity that originate in the eyestalk ganglia was present in the four crayfish but not in any other species. These neurons project mainly into the lateral soma clusters (cluster 10) comprising the somata of ascending olfactory projection neurons and innervate very sparsely the medial soma clusters (clusters 9 and 11) containing the somata of local interneurons. In the innervation pattern of the lateral cluster, the dopamine-immunoreactive neurons showed large species-specific differences. The other system comprises a pair of giant neurons with substance P-like immunoreactivity. These neurons have somata in the median protocerebrum of the central brain and major projections into the lateral clusters and the core of the olfactory lobes, the neuropils that are the first synaptic relay in the central olfactory pathway of decapods; minor arborizations are present in the medial clusters. The system of substance P-immunoreactive giant neurons was present and of great morphological similarity in all studied species. Only in one species, the shrimp Macrobrachium rosenbergii, evidence for co-localization of FMRFamide-like with substance P-like immunoreactivity in these neurons was obtained. These and previously collected data indicate that the centrifugal neurons with dopamine-like immunoreactivity may be associated with the presence of an accessory lobe, a second-order neuropil that receives input from the olfactory lobe and only occurs in spiny lobsters, clawed lobsters and crayfish. The pair of centrifugal giant neurons with substance P-like immunoreactivity, on the other hand, appears to be a constitutive component of the decapod crustacean brain that most likely is functionally associated with the olfactory lobe. Both systems apparently exert modulatory functions on olfactory information processing by preferentially targeting the somata of the projection neurons. Thus, in the olfactory projection neurons, the somata seem to be more directly involved in information processing than in most other neurons of the arthropod CNS.

Animals↗

PubMed1989

Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography. I. Design of the apparatus and analysis of acceleration.

A novel design of the cross-axis synchronous flow-through coil planet centrifuge is introduced. The apparatus holds a pair of large coil holders symmetrically, one on each side of the rotary frame, at a lateral position 12.5 cm from the center of the holder shaft held 10 cm from the centrifuge axis. Mathematical analysis of acceleration generated by the planetary motion of the apparatus revealed a unique centrifugal force field which promises high retention of the stationary phase in the multilayer coil to perform efficient preparative-scale counter-current chromatography.

Chromatography↗

PubMed1989

Centrifugal counter-current partition chromatography with helical coil rotor. Simplified counter-current chromatography with a rotating face-seal.

A centrifugal counter-current partition chromatograph has been developed and tested in order to simplify earlier counter-current chromatographic (CCC) procedures. It includes a helical coil rotor and a rotating face-seal. The rotor is designed to be adapted in an ordinary laboratory centrifuge for toroidal coil CCC. Twisting between the inlet and outlet tubing is avoided by using the rotating seal in the rotor. The seal is placed between the rotor, on which helical coils are mounted, and a newly designed centrifuge lid. This chromatographic rotor, rotating simply around its own axis, has simplified a previous CCC device in which a coil planet mechanism is used to avoid tube twisting whilst retaining the capability for chromatographic separations. Results for separations of nystatin, dinitrophenylamino acids and Poly I:C were comparable to those obtained by liquid chromatography and the previous CCC procedure.

Amino Acids↗

PubMed2002

Cause of kinematic differences during centrifugal and centripetal saccades.

Measurements of eye movements have shown that centrifugal movements (i.e. away from the primary position) have a lower maximum velocity and a longer duration than centripetal movements (i.e. toward the primary position) of the same size. In 1988 Pelisson proposed that these kinematic differences might be caused by differences in the neural command signals, oculomotor mechanics or a combination of the two. By using the result of muscle force measurements that were made in recent years (Orbit 1.8 Gaze mechanics simulation, Eidactics, San Francisco, 1999) we simulated the muscle forces during centrifugal and centripetal saccades. Based on these simulations we show that the cause of the kinematic differences between the centrifugal and centripetal saccades is the non-linear force-velocity relationship (i.e. muscle viscosity) of the muscles.

Biomechanical Phenomena↗

PubMed1999

Functional anatomy of the avian centrifugal visual system.

Although first described over a century ago, the centrifugal visual system (CVS) projecting to the retina still remains somewhat of an enigma with regard to its functional role in visually-guided behavior. The highly developed avian CVS has been the most extensively investigated and the anatomical organization of its two component centrifugal structures, the n. isthmo-opticus (NIO) and ectopic neurons (EN), including its afferent brainstem projections is reviewed. The results of double-labeling studies combining axonal tracing techniques and immunohistofluorescence have demonstrated GABA immunoreactivity (-ir) of interneurons within the neuropilar zone of the NIO, choline acetyltransferase (ChAT)-ir and nitric oxide synthase (NOS)-ir in the centrifugal cells of the NIO and EN as well as in the afferent projection neurons of layers 9/10 of the optic tectum. The data are discussed in terms of neurochemical and excitatory/inhibitory mechanisms within the different components of the avian CVS in relation to hypotheses which have implicated this system in visual attention and ground-feeding behavior.

Animals↗

PubMed2002

Centrifugal neurons of the octopus optic lobe cortex are immunopositive for calcitonin gene-related peptide.

Distribution of calcitonin gene-related peptide (CGRP)-like substance in the optic lobe cortex and retina of the octopus was examined immunohistochemically. Wheat germ agglutinin (WGA), a retrograde-transporting marker, was also used to label the centrifugal neurons. CGRP-immunoreactive (CGRP-IR) somata were seen in the inner granular cell layer, but not in the outer granular cell layer or the retina. CGRP-IR fibers were seen not only in the optic lobe cortex, but also in the retinal nerve plexus. Retrogradely labeled somata were seen in the inner granular cell layer, but not in the outer granular cell layer. Immunohistochemical double staining indicated that WGA-labeled centrifugal neurons were immunopositive for CGRP. These results suggested that the centrifugal neurons in the octopus optic lobe cortex are CGRP-like peptide-containing neurons, and that the peptide may modulate photoreceptor cell functions.

Animals↗

PubMed1998

Initial experience with the Nikkiso centrifugal pump during thoracoabdominal aortic aneurysm repair.

PURPOSE: Several centers use atriodistal bypass (ADB) as a protective adjunct against distal ischemia during extensive thoracoabdominal aortic aneurysm (TAAA) repair. Most current ADB circuits use indirect-drive centrifugal pumps. The purpose of this report is to describe our initial clinical experience with the Nikkiso pump, a more compact direct-drive centrifugal pump recently developed at Baylor, for ADB during TAAA repair. METHODS: The Nikkiso pump was used for ADB perfusion in 10 consecutive patients during graft repair of TAAAs (six Crawford extent I and four extent II). Two patients had aortic dissection. In the four patients who had extent II repairs, selective renal and visceral perfusion was also performed with the Nikkiso pump. RESULTS: No mechanical pump malfunctions or adverse events related to the device occurred. All 10 patients survived and were discharged from the hospital. No patient had paraplegia after surgery. Two patients had delayed lower extremity weakness after undergoing extent I repairs; both recovered and were ambulating at the time of discharge. No complications were associated with bleeding or cerebral, respiratory, renal, or hepatic function. CONCLUSIONS: Our initial experience with the Nikkiso centrifugal pump during TAAA repair demonstrated excellent pump function that provided sufficient flow for both distal aortic and selective organ perfusion. The prevention of permanent spinal cord injury and distal organ failure was successful in this group.

Aged↗

PubMed1997

Surgery of descending thoracic aortic aneurysms with centrifugal pump support.

Fifty-five patients with descending thoracic aortic aneurysms were operated upon between October 1987 and October 1994. All patients were supported by a centrifugal pump during operation. The mean(s.d.) duration of cross-clamping was 39(13) min. In order to evaluate the efficacy of the centrifugal pump, haemodynamic and metabolic measurements were made on four occasions (before cross-clamping, immediately after cross-clamping and before cross-clamp removal) and again after cross-clamp removal. The haemodynamic data remained stable throughout the procedure: central venous pressure (15(4.6) versus 16(4.8) versus 16(4.6) versus 15(4.6) mmHg; P = n.s.), pulmonary artery pressure (25(6.2) versus 24(5.1) versus 22(5.3) versus 23(4.4) mmHg; P = n.s.), radial systolic pressure (119(19.9) versus 116(25.2) versus 111(25.9) versus 111(20.7) mmHg; P = n.s.) and heart rate (75(12.6) versus 77(14) versus 76(15.6) versus 78(16) beats/min; P = n.s.). The acid-base status deteriorated slowly during surgery. Values before and after cross-clamping were: pH (7.42 (0.04) versus 7.37(0.06); P < 0.05), base excess (-0.67(2.20) versus -3.70(2.50); P < 0.05) and bicarbonates (24(8.9) versus 20(1.9); P < 0.05). The cerebrospinal fluid pressure remained constant: 20(5.7) versus 19(5.9) versus 18(5) versus 19(5) mmHg; P = n.s. Renal function, measured before, and at 1, 3 and 7 days after the operation also remained stable (creatinine: 1.1(0.4) versus 1.2(0.4) versus 1.2(0.4) versus 1.2(0.4); P = n.s.; blood urea nitrogen: 46(18.7) versus 46(18.6) versus 51(24.9) versus 55(27.9); P = n.s.). Step-wise multiple linear regression comparing cerebrospinal fluid pressure against haemodynamic and metabolic data showed that during aortic cross-clamping there was a significant relationship between central venous pressure (P < 0.0013) and arterial pH (P < 0.0148), while before and after cross-clamping multivariate analysis showed a relationship only between central venous pressure and cerebrospinal fluid pressure (P < 0.0035). The results confirm that centrifugal pump support is effective in stabilizing haemodynamics and protecting the kidney during thoracoabdominal aneurysm repair.

Acid-Base Equilibrium↗

PubMedDate not supplied

Centrifugal innervation of the rat retina.

Centrifugal fibers innervating the retina have been shown in all classes of vertebrate, except for mammals where conventional tract-tracing methods have not been able to unmistakably demonstrate their existence. In a previous study, a unilateral, intravitreal injection of 5,7-dihydroxytryptamine was used to reveal indoleamine-accumulating centrifugal fibers which were visualized by an immunoreaction against serotonin. In the present study, I employed a modification of this method to stain retinopetal neurons in the rat. Terminals were located preferentially in the outer retina; labeled fibers could be traced back along an ipsilateral pathway to somata in the dorso-caudal portions of the chiasm or the medio-lateral preoptic area, and thence towards the suprachiasmatic nuclei. The unique beaded appearance of the fibers distinguishes them from retinal ganglion cell axons. The labeling of central cell bodies strongly suggests that they possess terminals in the retina. Thus, at least some mammalian retinas receive centrifugal innervation. This indoleamine-accumulating retinopetal pathway may be involved in retinal melatonin synthesis, coordination of circadian rhythms, and interocular phenomena.

5,7-Dihydroxytryptamine↗

PubMed2004

Investigation into the applicability of the centrifugal microfluidics platform for the development of protein-ligand binding assays incorporating enhanced green fluorescent protein as a fluorescent reporter.

The incorporation of a protein-ligand binding assay into a centrifugal microfluidics platform is described. The platform itself is a disc-shaped polymer substrate, upon which a series of microfluidic channels and reservoirs have been machined. Centrifugal microfluidics platforms require no internal moving parts, and fluid propulsion is achieved solely through rotation of the disc. Fluid flow is controlled by passive valves, the opening of which is dependent on the angular frequency of the rotating platform, the channel dimensions, and the physical properties of the fluid. To evaluate the effectiveness of incorporating a protein-based assay onto the centrifugal microfluidics analytical platform, a class-selective, homogeneous assay for the detection of phenothiazine antidepressants was employed. This class of drugs is known to bind to calmodulin, a calcium binding protein. Specifically, a fusion protein between calmodulin and enhanced green fluorescent protein was utilized. Calmodulin undergoes a conformational change upon binding to phenothiazines that alters the fluorescence properties of the attached fluorescent protein, which can be correlated to the concentration of the drug present. Another important aspect of this work was to study the efficacy of the platform to perform reconstitution assays. To do this, the biological reagent was dried on the platform and rehydrated to carry out the assay. The ability to prealiquot reagents on the platform should enhance its versatility and portability. The integration of protein-based assays in this platform should be useful in the design of analytical systems for high-throughput screening of pharmaceuticals and clinical diagnostics.

Antidepressive Agents↗

PubMed1979

High concentration active enzyme centrifugation: analysis of active polymeric forms at up to 10 000-fold higher concentrations than with conventional methods.

This paper describes the theoretical basis, experimental technique, and experimental evaluation of a new method of analysis called "high concentration active enzyme centrifugation". It extends by up to four orders of magnitude the upper concentration limits at which the technique of "active enzyme centrifugation" can be used for analysis of enzyme structure. This new theory is largely based on certain properties of Gaussian curves which we have described in previous publications [Wei, G.J., & Deal, W.C., Jr. (1976) Anal. Biochem. 75, 113-121; Anal. Biochem. (1978) 87, 433-446]. One of the most important aspects of this development is that it extends the concentration range upward so that experiments can be performed on enzymes in the active polymeric forms corresponding to their in vivo states. Furthermore, this expansion includes the range in which most enzymes go through all their association-dissociation transitions from one polymeric form to another. Hence, the method can be used to define the various concentration-dependent transitions and also to ascertain which of the various polymeric forms of an enzyme are active, under various conditions. This method also retains the many favorable characteristics inherent in the active enzyme centrifugation technique. In studies with lactate dehydrogenase, the results from this method of band sedimentation were identical within experimental error (about 1.5%) with results from conventional boundary sedimentation velocity studies.

Animals↗

PubMed2002

Binding of hemoglobin to red cell membranes with eosin-5-maleimide-labeled band 3: analysis of centrifugation and fluorescence data.

We have studied the binding of hemoglobin to the red cell membrane by centrifugation and fluorescence methods. The intact red cell was labeled with eosin-5-maleimide (EM), which specifically reacts with lysine 430 of band 3. Even though this residue is not part of the cytoplasmic domain of band 3 (cdb3) associated with hemoglobin binding, fluorescence quenching was observed when hemoglobin bound to inside-out vesicles (IOVs). The use of fluorescence quenching to measure band 3 binding was quantitatively compared with the binding determined by centrifugation, which measures binding to band 3 and non-band 3 sites. For the centrifugation it was necessary to include the non-band 3 association constants determined from chymotrypsin-treated IOVs. The binding of hemoglobin to band 3 was interpreted in terms of the binding of two hemoglobin tetramers to each band 3 dimer. An anticooperative interaction associated with the conformational change produced when hemoglobin binds results in a 2.8-fold decrease in the intrinsic constant of (1.54 +/- 0.25) x 10(7) M(-1) for the binding of the second hemoglobin molecule. From the changes in lifetime produced by binding the first and second hemoglobin molecules, it was possible to show that the conformational change associated with binding the second hemoglobin molecule results in a decrease of the heme-eosin distance from 47.90 to 44.78 A. Reaction of cyanate with the alpha-amino group of hemoglobin (HbOCN) is shown to produce a very dramatic decrease in the binding of hemoglobin to both the band 3 and non-band 3 sites. The intrinsic constant for binding the first hemoglobin molecule to band 3 decreases by a factor of 29 to (5.34 +/- 0.15) x 10(5) M(-1). The anticooperative interaction is greater with the intrinsic constant decreasing by a factor of 3.8 for the binding of the second hemoglobin tetramer to band 3. In addition, the nature of the conformational change produced by binding hemoglobin is very different with the second HbOCN increasing the heme-eosin distance to 55.99 A. The utilization of eosin-5-maleimide-reacted red cell membrane to study hemoglobin binding makes it possible to directly study the binding to band 3. At the same time a sensitive probe of the conformational changes, which occur when hemoglobin binds to band 3, is provided.

Anion Exchange Protein 1, Erythrocyte↗