Is the interaction of CNP with tubulin and microtubules required for process extension in oligodendrocytes?

The STOP protein (stable tubule-only polypeptide) is a calmodulin-regulated protein which associates with microtubules and induces cold stabilization. There are different isoforms of this protein that arise from alternative splicing of STOP mRNA. Neurons express two major variants N-STOP (125 kDa) and E-STOP (84 kDa). NIH 3T3 fibroblasts contain a major F-STOP isoform (42 kDa) and two minor STOP variants (48 and 89 kDa). Previously, we demonstrated the presence of N-STOP in the cytoskeleton associated with myelin isolated from animals injected with apotransferrin. Since this protein was only described as a neuronal protein we decided to further investigate the expression of this protein in oligodendrocyte cultures. The analysis of the STOP protein expression in oligodendrocyte shows that STOP protein is expressed in the soma and processes of oligodendrocyte precursors, as well as in immature and mature oligodendroglial cells. In addition, we found that MBP shows a high degree of colocalization with STOP protein. By Western blot analysis, it was found that these cells express a major STOP variant (89 kDa). When the cultures were exposed to cold temperature we found that STOP protein associates with microtubules and induces microtubule cold stabilization. Under these experimental conditions, we found that MBP associates with microtubules too, and maintains its colocalization with STOP protein. At present, we are doing new assays directed to further characterize STOP (89 kDa) protein and to elucidate how this protein participates in the formation of myelin by oligodendrocytes.     

Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Re-examination of the Post-translational Arginylated Protein of 125-kD Initially Identified as N-STOP

Post-translational modification of proteins is a complex mechanism by which cells regulate protein activities. One post-translational modification is the incorporation of arginine into the NH2-terminus of proteins. It has been hypothesized that in rat brain extracts, one of the proteins modified by this reaction is the microtubule-associated protein Neuronal Stable Tubule Only Polypeptide (N-STOP). This was inferred from its electrophoretic mobility (125 kD) and because it was immunoprecipitated with a monoclonal antibody against the N-STOP. However, this hypothesis is not supported by our recent results. Herein, we found that rat N-STOP interacts with Ca(2+)-calmodulin, whereas the 125-kD [14C]-arginylated protein does not. The 125-kD [14C]-arginylated protein from rat brain is separated from the N-STOP by two-dimensional electrophoresis, and it is not recognized by a STOP monoclonal antibody. Mouse brain contains N-STOP, which migrates as a protein of 116 kD and could not be labeled by the post-translational incorporation of [14C]-arginine. The 125-kD [14C]-arginylated protein appears in wild-type as well as in STOP knock out mice. Based on these results, we conclude that the 125-kD arginylated protein is different from N-STOP.     

STOP Proteins are Responsible for the High Degree of Microtubule Stabilization Observed in Neuronal Cells

Neuronal differentiation and function require extensive stabilization of the microtubule cytoskeleton. Neurons contain a large proportion of microtubules that resist the cold and depolymerizing drugs and exhibit slow subunit turnover. The origin of this stabilization is unclear. Here we have examined the role of STOP, a calmodulin-regulated protein previously isolated from cold-stable brain microtubules. We find that neuronal cells express increasing levels of STOP and of STOP variants during differentiation. These STOP proteins are associated with a large proportion of microtubules in neuronal cells, and are concentrated on cold-stable, drug-resistant, and long-lived polymers. STOP inhibition abolishes microtubule cold and drug stability in established neurites and impairs neurite formation. Thus, STOP proteins are responsible for microtubule stabilization in neurons, and are apparently required for normal neurite formation.     

Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin

Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90–177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90–177) to microtubules with a 1:1 MAP6(90–177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90–177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca²⁺-calmodulin competes with microtubules for MAP6(90–177) binding and that the binding mode of MAP6(90–177) to microtubules and Ca²⁺-calmodulin involves a common stretch of amino acid residues on the MAP6(90–177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca²⁺-calmodulin.     

Specific association of STOP protein with microtubules in vitro and with stable microtubules in mitotic spindles of cultured cells.

STOP (Stable Tubule Only Polypeptide) is a neuronal microtubule associated protein of 145 kd that stabilizes microtubules indefinitely to in vitro disassembly induced by cold temperature, millimolar calcium or by drugs. We have produced monoclonal antibodies against STOP. Using an antibody affinity column, we have produced a homogeneously pure 145 kd protein which has STOP activity as defined by its ability to induce cold stability and resistance to dilution induced disassembly in microtubules in vitro. Western blot analysis, using a specific monoclonal antibody, demonstrates that STOP recycles quantitatively with microtubules through three assembly cycles in vitro. Immunofluorescence analysis demonstrates that STOP is specifically associated with microtubules of mitotic spindles in neuronal cells. Further, and most interestingly, STOP at physiological temperature appears to be preferentially distributed on the distinct microtubule subpopulations that display cold stability; kinetochore-to-pole microtubules and telophase midbody microtubules. The observed distribution suggests that STOP induces the observed cold stability of these microtubule subpopulations in vivo.     

Monoclonal antibody to microtubule-associated STOP protein: affinity purification of neuronal STOP activity and comparison of antigen with activity in neuronal and nonneuronal cell extracts

Microtubules, ordinarily cold-labile structures, are made entirely resistant to cold temperature by the presence of substoichiometric amounts of STOP (stable tubule only polypeptide), a microtubule-associated protein. We have produced a monoclonal antibody which specifically recognizes a 145-kDa protein previously implicated in STOP activity in rat brain extracts. An antibody affinity column removes both the 145-kDa protein and STOP activity from solution. A urea eluate from the affinity column contains the 145-kDa protein and exhibits substantial STOP activity. We conclude the 145-kDa protein accounts for all measurable STOP activity in rat neuronal extracts. For this work, we have developed an assay of microtubule cold stability which is generally applicable to the detection of STOP activity in various tissues. Using this assay, we show STOP activity is most abundant in neuronal tissue but is detectable in all tissues tested, with the exception of heart muscle. In all tissues that we have examined, STOP activity elutes as a single peak from heparin affinity columns, and in common with brain STOP, all activity is Ca2+-calmodulin sensitive. The monoclonal antibody recognizes the 145-kDa STOP in rat neuronal extracts but reacts with no protein in active fractions from other tissue. A similar, but not identical, analogue of brain STOP thus appears to be widespread in mammalian tissues.     

STOP proteins

Microtubules assembled from pure tubulin in vitro are labile, rapidly depolymerized upon exposure to the cold. In contrast, in a number of cell types, cytoplasmic microtubules are stable, resistant to prolonged cold exposure. During the past years, the molecular basis of this microtubule stabilization in cells has been elucidated. Cold stability is due to polymer association with different variants of a calmodulin-regulated protein, STOP protein. The dynamic and hence the physiological consequences of STOP association with microtubules vary in different tissues. In neurons, STOP seems almost permanently associated with microtubules. STOP is apparently a major determinant of microtubule turnover in such cells and is required for normal neuronal differentiation. In cycling cells, only minor amounts of STOP are associated with interphase microtubules and STOP does not measurably affects microtubule dynamics. However, STOP is associated with mitotic microtubules in the spindle. Recent results indicate that such an association could be vital for meiosis and for the long-term fidelity of the mitotic process.     

Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes (Mithieux, G., Audebet, C., and Rousset, B. (1988) Biochim. Biophys. Acta 969, 121-130), a process which is inhibited by ATP (Mithieux, G., and Rousset, B. (1988) Biochim. Biophys. Acta 971, 29-37). Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells.     

Characterization of a 60-kDa cell surface-associated transforming growth factor-β binding protein that can interfere with transforming growth factor-β receptor binding

We have characterized a 60-kDa transforming growth factor-β (TGF-β) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross-linking of cell surface proteins by using ¹²⁵I-TGF-β1. Binding of ¹²⁵I-TGF-β1 to the 60-kDa protein was competed by an excess of unlabeled TGF-β1 but not by TGF-β2, TGF-β3, activin, or osteogenic protein-1 (OP-1), also termed bone morphogenetic protein-7 (BMP-7). In addition, no binding of ¹²⁵I-TGF-β2 and ¹²⁵I-TGF-β3 to the 60-kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60-kDa TGF-β binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF-β superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 μg/ml heparin, or 10% glycerol caused a release of the 60-kDa protein from the cell surface. In addition, we found that the previously described TGF-β type IV receptor on GH3 cells, which does not form a heteromeric complex with TGF-β receptors, could be released from the cell surface by these same treatments. This suggests that the 60-kDa protein and the similarly sized TGF-β type IV receptor are related proteins. The eluted 60-kDa LNCaP protein was shown to interfere with the binding of TGF-β to the TGF-β receptors. Thus, the cell surface-associated 60-kDa TGF-β binding protein may play a role in regulating TGF-β binding to TGF-β receptors. J. Cell. Physiol. 173:447–459, 1997. © 1997 Wiley-Liss, Inc.     

The 72-kDa Microtubule-Associated Protein from Porcine Brain

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

DMAP-85: A τ-Like Protein from Drosophila melanogaster Larvae

Abstract: Microtubule-associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol-polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85-kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85-kDa protein bound specifically to an affinity column of Sepharose-βII-(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII-tubulin. Affinity-purified 85-kDa protein enhanced microtubule assembly in a concentration-dependent manner. This effect was significantly decreased by the presence of the βII-(422–434) peptide in the assembly assays, thus confirming the specificity of the 85-kDa protein interaction with the C-terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti-τ antibodies, including sequence-specific probes that recognize repeated microtubule-binding motifs on τ, MAP-2, and MAP-4 and specific N-terminal sequences of τ, cross-reacted with the 85-kDa protein from Drosophila larvae. These results suggest that τ and Drosophila 85-kDa protein share common functional and structural epitopes. We have named this protein as DMAP-85 for Drosophila MAP. The finding on a Drosophila protein with functional homology and structural similarities to mammalian τ opens new perspectives to understand the cellular roles of MAPs.     

Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when ¹²⁵I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Phosphorylation of microtubule-associated protein STOP by calmodulin kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.     

Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

Background

STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis.

Methodology/Principal Findings

Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice.

Conclusions/Significance

In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.     

Protein Methylation in Cerebellar Synaptosomes

Abstract: Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21–26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21–26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21–26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21–26-kDa family was identified as GTP-binding proteins by [α-³²P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [α-³²P]GTP. When synaptosomes were screened for putative methyltransferases by S -adenosyl-L-[ methyl -³H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.     

Covalent Cross-Linking of Radiolabeled N-Formylated Hexapeptide to its Specific Receptor on Rat and Human Neutrophils: Evidence for a ligand induced complex formation

Abstract

The structure of the rat and human neutrophil receptor for N-formylated chemotactic peptides was characterized using ¹²⁵I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys hexapeptide as a ligand and an affinity cross-linking technique. ¹²⁵I-hexapeptide bound to purified rat peritoneal neutrophils was time, temperature and pH-dependent. The average receptor number per cell was about 67.000 and díssociation constant (K_d) 0.41 nM. Formyl-MLLP, fMLP, fNLP, were 750%, 15%, 8.6% respectively and Boc-MLP, Boc-NLP, and MLP 0.6% as potent as the hexapeptide in inhibiting the binding of ¹²⁵I-hexapeptide to rat neutrophils. The same correlation was found between these peptides in their potency to induce chemotaxis. This indicates that the rat neutrophil chemotactic receptor is like human receptor also a highly stereoselective and requires a N-formylated ligand for high affinity binding. Affinity cross-linking of ¹²⁵I-hexapeptide to rat and human neutrophil chemotactic receptor with glutaraldehyde revealed on SDS-PAGE a 85-kDa and 62-kDa major complex and a 170-kDa and 120-kDa minor complex, respectively. The 120-kDa complex was absent in human neutrophils if the cells were treated with glutaraldehyde prior to cross-linking of ¹²⁵I-hexapeptide to its receptor. Likewise, the larger complex was absent if neutrophils were exposed to heterelogous ligand (C5a) prior to glutaraldehyde treatment and cross-linking of ¹²⁵I-hexapeptide to its specific receptor. These results demonstrate that the rat neutrophils possess a functional high-affinity receptor for N-formylated chemotactic peptides and that the size of the monomeric receptor is 85-kDa and about 23-kDa larger than that of the human receptor. Upon homologous ligand binding the receptor seems to form a larger complex.     

High concentrations of STOP protein induce a microtubule super-stable state

We have previously shown that mammalian brain crude extracts contained two classes of stable microtubules: "cold stable" and "super-stable" microtubules. We now find that both species are generated by a single protein factor (STOP protein) in a dose dependent manner. These results show that STOP protein action can be extreme, inducing resistance to -80 degrees C or to sonication and that no other factor seems to be required to account for the various subclasses of highly stable microtubules in brain. Finally, the rapid procedure described for the preparation of purified "super-stable microtubules" should be useful for the obtention of fractions with high STOP protein activity.     

Denaturation of Either Manduca sexta Aminopeptidase N or Bacillus thuringiensis Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions

The effect of polypeptide denaturation of Bacillus thuringiensis Cry1A toxins or purified Manduca sexta 120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with ¹²⁵I-labeled Cry1Ac, Cry1Ac mutant ⁵⁰⁹QNR-AAA⁵¹¹ (QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the N-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both ¹²⁵I-Cry1Ac and ¹²⁵I-QNR-AAA bound to 210- and 120-kDa proteins from M. sexta brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots ¹²⁵I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment, ¹²⁵I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA. ¹²⁵I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured ¹²⁵I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured) ¹²⁵I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.