Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules

The 4-kDa C-terminal domain of both tubulin subunits plays a major role in the regulation of microtubule assembly [Serrano et al. (1984) Biochemistry 23, 4675]. Controlled proteolysis of tubulin with subtilisin produces the selective cleavage of this 4-kDa moiety from alpha- and beta-tubulin with a concomitant enhancement of the assembly. Here we show that gradual removal of the last six to eight amino acid residues of the C-terminal region of alpha and beta subunits by an exopeptidase, carboxypeptidase Y, produces a modified protein (C-tubulin) without relieving the modulatory effect of the C-terminal domain and the usual need of MAPs for microtubule assembly. Actually, treatment with this proteolytic enzyme did not change tubulin assembly as promoted by either MAP-2, taxol, MgCl2, dimethyl sulfoxide, or glycerol. The critical concentration for the assembly of C-tubulin remained the same as that for the unmodified tubulin control. Microtubule-associated proteins MAP-2 and tau incorporated into C-tubulin polymers. Clearly, pure C-tubulin did not assemble in the absence of MAPs or without addition of assembly-promoting compounds. However, proteolysis with the exopeptidase induced changes in tubulin conformation as assessed by biophysical methods and double-limited proteolysis. The cleavage with subtilisin after carboxypeptidase digestion did not result in enhancement of the assembly to the levels observed after the treatment of native tubulin with subtilisin. Interestingly, Ca2+ ions affected neither C-tubulin assembly nor depolymerized microtubules assembled from C-tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a Ca2+-protease resistant high Mr microtubule binding protein from mammalian brain: Characterization of properties partially expected for a dynein-like molecule

Summary A novel microtubule binding protein was isolated from cell extracts of pig brain after selective destruction of high Mr MAPs by Ca²⁺-activation of endogenous proteases. The protein had an apparent Mr of 300,000, but several criteria, including peptide maps, immunological crossreactivities, resistance to Ca²⁺-activated proteolysis, and inability to induce microtubule assembly, distinguished this protein from the major high Mr microtubule associated proteins MAP-1 and MAP-2. Certain molecular properties of the protein resembled those of dynein: its size under denaturing conditions, its crossreactivity with antibodies toTetrahymena dynein, the ATP-dependence of its microtubule binding, the erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA)—sensitivity of its ATPase activity and its resistance to Ca²⁺-activated proteolysis. However, in peptide maps and the insensitivity of its ATPase activity to vanadate the protein could be differentiated fromTetrahymena dynein heavy chain polypeptides. Based on the properties characterized so far, the protein seems qualified to function as a mechanochemical enzyme in the cytoplasm of mammalian brain cells and could represent a cytoplasmic dynein variant.Abbreviations EGTA ethylene glycol bis([3-aminoethylether)N,N,N',N'-tetraacetic acid - MAP microtubule associated protein - Mr molecular weight - SDS sodium dodecyl sulfate     

Activation of H-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease

Background

Glucose induces H⁺-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H⁺-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity.

Methods

We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H⁺-ATPase activation after glucose treatment.

Results

We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35–40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H⁺-ATPase complex did not undergo dissociation, and H⁺-ATPase activation was significantly delayed.

Conclusion

Our findings indicate that the mechanism of H⁺-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H⁺-ATPase complex dissociation and the activation of the enzyme.

General significance

Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H⁺-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H⁺-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.     

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule-associated protein 2 (MAP-2). Activation of the N -methyl- d -aspartate (NMDA) receptor dephosphorylates MAP-2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP-2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP-2. Also, the amount of MAP-2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP-2 was decreased by ∼75% 1-2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP-2 was prevented completely by injection of 2 mg/kg MK-801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l -Carnitine, which protects rats against ammonia toxicity, also prevented MAP-2 degradation. Because activation of the NMDA receptor increases [Ca²⁺]_i, we determined whether rat brain contains a Ca²⁺-dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca²⁺-dependent protease that degrades MAP-2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca²⁺]_i, which activates calpain I. This, in turn, selectively degrades MAP-2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.     

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.     

Identification and spatial arrangement of high molecular weight proteins (Mr 300 000-330 000) co-assembling with microtubules from a cultured cell line (rat glioma C6)

The distribution of three high molecular weight proteins, MAP-1 (Mr 330 000), MAP-2 (Mr 300 000) and plectin (Mr 300 000) in various fractions obtained in cycles of temperature-dependent polymerization/depolymerization of microtubules from rat glioma C6 cells was studied. Using gel electrophoresis and immunoautoradiography/immunoblotting all three proteins were found to codistribute only partially with tubulin because considerable parts remained in the cold-insoluble fractions. Moreover, the proteins, particularly MAPs, were proteolytically degraded during cycling. By contrast, when microtubules were polymerized with taxol after isotonic cell lysis a considerable enrichment of MAP-1 and MAP-2 was achieved; again, plectin co-distributed only partially. In this procedure too, MAPs, especially MAP-2, were found to be highly subject to proteolysis, unless free Ca2+-ions were rigorously avoided. Proteolytic fragments generated from MAP-2 were of similar size independent of whether temperature- or taxol-dependent polymerization procedures were used, suggesting the occurrence of a MAP-2-specific protease. When the spatial arrangement of the high Mr proteins on taxol-polymerized C6 cell microtubules was directly visualized using gold-immunoelectron microscopy, a periodical, apparently helical, decoration of microtubules was found for MAP-1 and MAP-2; plectin was irregularly arrayed. A predominantly helical arrangement of both MAPs was demonstrated also for microtubules reconstituted from mammalian brain.     

Numerous phosphates of microtubule-associated protein 2 in living rat brain

Microtubule-associated protein 2 (MAP 2) purified from microwave-irradiated rat head contained about 46 esterified phosphates (mole/mol), which were not bound covalently to lipids and did not assemble with microtubules. After some phosphates were released by calf intestinal alkaline phosphatase, the phosphate content of MAP-2 decreased to 16 mol of phosphate and the protein assembled in vitro. MAP-2 purified after microtubule assembly cycles and also the cytosolic heat-stable fraction without assembly cycles had 10 mol of phosphate, and both assembled with microtubules. The MAP-2 with 46 phosphates and that with 10 had different pI in isoelectric focusing, but the components, MAP-2a and -2b, were always near each other. In high-pressure liquid chromatography, MAP-2 containing 46 mol of phosphate appeared after that 10 mol of phosphate. Phosphoserine, phosphothreonine, and phosphotyrosine were recovered from tryptic digestion of MAP-2 with 46 mol of phosphate. These findings suggest that two kinds of MAP-2, one with 46 phosphates and not bound to tubulin and the other with 10-16 phosphates and bound to tubulin, are present in the living rat brain.     

Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells.

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.     

Purification of tropomyosin, paramyosin, actin, tubulin, troponin and kinases for chemiproteomics and its application to different scientific fields

Background

p-aminobenzamidine (p-ABA) is used as a ligand in the purification of many serine proteases and in their removal from heterogeneous samples. Moreover, p-ABA has a potent ability to bind Ca²⁺-binding proteins. The binding ability and use of p-ABA in purification processes is still not fully understood.

Methodology/Principal Findings

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of the panallergenic proteins tropomyosin and paramyosin, as well as actin, tubulin, troponin and several kinases and annexins, with variable specificity depending on the tissue source and slight modifications to the purification process. The high affinity of p-ABA to tropomyosin, paramyosin, actin, troponin and myosin is calcium-dependent, since calcium regulates the function of these proteins. In addition, p-ABA probably simulates phosphorylated serine and therefore purified appropriate kinases. Because p-ABA binds to calcium-dependent proteins, and probably those with binding sites containing serine, it is not a suitable inhibitor of proteolysis during the purification of such proteins. p-ABA is widely used to inhibit proteases during protein purification processes, but it is used in columns here to purify non-protease proteins. Two strategies were applied; the first was the inactivation of proteases that were not of interest using protease inhibitors. The second strategy employed was the use of a Ca²⁺ wash solution to remove calcium-dependent proteins. The removal of calcium-dependent proteins from rabbit hind muscle pointed out even more selective purification. It is possible to obtain two purified samples: a) calcium dependent proteins and b) calcium independent proteins. Moreover, p-ABA may be useful as a model to study processes involving the phosphorylation of serine.

Conclusion

A p-Aminobenzamidine (p-ABA) ligand enabled the purification of non-protease proteins, with variable specificity depending on the tissue source and slight modifications to the purification process. The method is applicable to various scientific branches, but is especially practical for medicinal applications.     

Tubulin must be acetylated in order to form a complex with membrane Na+,K+-ATPase and to inhibit its enzyme activity

In cells of neural and non-neural origin, tubulin forms a complex with plasma membrane Na⁺,K⁺-ATPase, resulting in inhibition of the enzyme activity. When cells are treated with 1 mM L-glutamate, the complex is dissociated and enzyme activity is restored. Now, we found that in CAD cells, ATPase is not activated by L-glutamate and tubulin/ATPase complex is not present in membranes. By investigating the causes for this characteristic, we found that tubulin must be acetylated in order to associate with ATPase and to inhibit its catalytic activity. In CAD cells, the acetylated tubulin isotype is absent. Treatment of CAD cells with deacetylase inhibitors (trichostatin A or tubacin) caused appearance of acetylated tubulin, formation of tubulin/ATPase complex, and reduction of membrane ATPase activity. In these treated cells, addition of 1 mM L-glutamate dissociated the complex and restored the enzyme activity. Cytosolic tubulin from trichostatin A-treated but not from non-treated cells inhibited ATPase activity. These findings indicate that the acetylated isotype of tubulin is required for interaction with membrane Na⁺,K⁺-ATPase and consequent inhibition of enzyme activity.     

Multiple Proteases Are Involved in Thymocyte Apoptosis

To investigate the involvement of proteases in apoptosis, rat thymocytes were treated with the glucocorticoid hormone methylprednisolone or the topoisomerase II inhibitor etoposide in the presence of selective substrate inhibitors of either interleukin-1β-converting enzyme (ICE), (Z-Val-Ala-Asp-chloromethylketone, VADcmk) or Ca²⁺-regulated serine protease (Suc-Ala-Ala-Pro-Phe-chloromethylketone, AAPFcmk). VADcmk protected from lamin proteolysis, chromatin fragmentation, cell shrinkage, and formation of apoptotic nuclei in both methylprednisolone- and etoposide-treated thymocytes when present during the initiation of the apoptotic process. AAPFcmk prevented lamin breakdown, chromatin fragmentation, and apoptotic morphological changes in thymocytes treated with methylprednisolone, but not with etoposide. Both MPS- and etoposide-treated thymocytes exhibited enhanced ICE-like protease activity which was maximal 1 h after treatment. This increase in proteolytic activity was blocked by VADcmk, but not AAPFcmk. Our findings suggest that ICE-like protease activity is critically involved in the early phase of both methylprednisolone- and etoposide-induced apoptosis in thymocytes, whereas the Ca²⁺-regulated serine protease is an obligatory component of the proteolytic cascade in methylprednisolone-induced apoptosis.     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

Cleavage of Bovine Brain Microtubule-Associated Protein-2 by Human Immunodeficiency Virus Proteinase

The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.     

Cyclic modulation of cross-linking interactions of microtubule-associated protein-2 with actin and microtubules by protein kinase FA

The ATP.Mg-dependent type-1 protein phosphatase activating factor (factor FA) was identified as a brain protein kinase that could phosphorylate microtubule-associated protein-2 (MAP-2) and thereby inhibit cross-linking interactions of MAP-2 with actin filaments and microtubules isolated from porcine brain. The phosphorylation sites were found to be equally located on both projection and microtubule-binding domains of MAP-2. Phosphoamino acid analysis revealed that the phosphorylation sites were on both serine and threonine residues, indicating that factor FA is a serine/threonine-specific MAP-2 kinase. Conversely, factor FA was further identified as a MAP-2 phosphatase activator that could promote the dephosphorylation of³²P-MAP-2 phosphorylated by factor FA itself and thereby potentiate cross-linking interactions of MAP-2 with actin and microtubules. Furthermore, the two opposing functions of factor FA can be selectively modulated in a reciprocal manner bypH change. For instance, alkalinepH could stimulate factor FA to work as a MAP-2 kinase but simultaneously block it to work as a MAP-2 phosphatase activator to potentiate the inhibition on the cross-linking interactions of MAP-2 with actin and microtubules. Taken together, the results provide initial evidence that a cyclic modulation of cross-linking interactions of MAP-2 with actin filaments and microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for rapid structural and functional regulation of neuronal cytoskeletal system.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

The effect of metal ions on the alkaline phosphatase of Rhizobium leguminosarum

The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg²⁺ and 1.9–5.1 g-atoms Zn²⁺ per mole of enzyme. In addition high concentrations of Mg²⁺ markedly stimulate the enzyme. The phosphatase is inhibited by Li⁺ and Na⁺ and stimulated by K⁺, Rb⁺ and Cs⁺, which suggests that the enzyme is K⁺ activated.     

Localization of Specific Epitopes on Human Microtubule-Associated Protein 2

Abstract: Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169–219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.     

In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.     

棉铃虫幼虫中肠主要蛋白酶活性的鉴定

根据棉铃虫Helicoverpa armigera(Hubner)中肠酶液对蛋白酶专性底物在不同pH下的水解作用,棉铃虫中肠的3种丝氨酸蛋白酶得到鉴定。它们是：强碱性类胰蛋白酶,水 解a-N-苯甲酰-DL-精氨酸-p-硝基苯胺的最适pH在10.50以上;弱碱性类胰蛋白酶,水解p-甲苯磺酰-L-精氨酸甲酯的最适pH为8.50～9.00;类胰凝乳蛋白酶, 水解N一苯甲酰-L-酪氨酸乙酯的最适pH亦为8.50-9.00。中肠总蛋白酶活性用偶 氮酪蛋白测定,最适pH亦在10.50以上。Ca²⁺对昆虫蛋白酶无影响,Mg²⁺仅对弱碱性类胰蛋白酶有激活作用。对苯甲基磺酰氟和甲基磺酰-L-赖氨酸氯甲基酮对弱碱性类胰蛋白酶的抑制作用较强,而对强碱性类胰蛋白酶的抑制作用较弱。甲基磺酰-L苯丙氨酸氯甲基酮除能抑制类胰凝乳蛋白酶外,还能激活弱碱性类胰蛋白酶。对牛胰蛋白酶有强抑制作用的卵粘蛋白抑制剂对昆虫蛋白酶却无抑制作用。大豆胰蛋白酶抑制剂对该虫的3种丝氨酸蛋白酶均有强的抑制作用。