Purification of a calcium-activated neutral proteinase from bovine brain

A calcium-activated neutral proteinase (CANP) resolved into three components has been partially purified from bovine brain. The method of isolation has resulted in 22,000, 7,100, and 8,000-fold purification for CANP I, II and III respectively. All three fractions require Ca2+ for activation. The characterization of the purified CANP I has shown that it is activated by 250 microM Ca2+ and the enzyme loses its activity when incubated in the presence of Ca2+ without substrate. Mg2+ is ineffective. The enzyme degrades neurofilament triplet proteins, tubulin and casein efficiently. The myelin basic protein is hydrolyzed after longer incubation. Bovine serum albumin and histones are unaffected. The enzyme is active at pH 5.5 to 9.0 with optimum between pH 7.5 and 8.5. It has a Km of 1.8 X 10(-7) M for the 69,000 dalton neurofilament protein. The enzyme is inhibited by sulphydryl blocking reagents and also by EGTA, leupeptin and E-64c. The SDS-PAGE analysis of the enzyme fractions has shown a major band at 66-68,000 daltons and two minor bands at 60,000 and 48-50,000 daltons for CANP I; a major band at 48-50,000 daltons and a minor band at 30-32,000 daltons for CANP II and a predominant doublet at 30-32,000 daltons with a minor band at 48-50,000 daltons for CANP III. The degradation of neurofilament proteins suggests that the CANP(s) may be involved in the turnover of these proteins.     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.     

Characterization of calcium-activated neutral protease (CANP)-associated protein kinase from bovine brain and its phosphorylation of neurofilaments

Calcium-activated neutral protease with low affinity for calcium (CANP II, Mr 76,000) can be purified to apparent homogeneity by casein affinity chromatography but contains cyclic-AMP dependent protein kinase activity. CANP II-associated kinase from bovine brain copurifies with protease activity through multiple chromatographic procedures but can be separated by cyclic-AMP affinity chromatography. Isolated protein kinase has subunits of Mr 80,000, 53,000 and 42,000. The kinase preferentially "autophosphorylates" CANP II, but histones, phosphorylase b and neurofilament proteins are also good substrates. The concentrations for half-maximal phosphorylation activity (Km) of cyclic-AMP, (32P)ATP and Mr 150,000 neurofilament protein substrate are 0.2, 6.0 and 0.5 microM, respectively. The specific activity of CANP II associated kinase in phosphorylating neurofilament proteins is intermediate between that of neurofilament- and MAPs 2-associated kinases.     

Preparative separation and amino acid composition of neurofilament triplet proteins

Abstract: Intact neurofilaments were isolated from bovine spinal cord white matter, washed by sedimentation in 0.1 m -NaCl, and extracted with 8 m -urea. Solubilized neurofilament triplet proteins of molecular weights approximately 68,000 (P68), 150,000 (P150), and 200,000 (P200) were purified by preparative electrophoresis, using an LKB 7900 Uniphor apparatus. The method provides for an enhanced yield of purified protein and has markedly reduced admixture of electrophoresed protein with acrylamide and associated protein contaminants. Amino acid compositions of the purified neurofilament triplet proteins are reported and compared.     

Characterization of mammalian neurofilament triplet proteins. Subunit stoichiometry and morphology of native and reconstituted filaments

The three major proteins of mammalian neurofilaments of molecular weights 179,000 (NF1), 129,000 (NF2), and 66,500 (NF3) have been purified to homogeneity by multiple anion-exchange and hydroxylapatite absorption chromatography in 8 M urea. Silver staining of polyacrylamide gels of the purified proteins show single bands. In order to gain further insight into the molecular organization of the neurofilament triplet proteins, the molar stoichiometries and morphologies of native and reconstituted filaments and those isolated from developing brain were studied. Denaturing polyacrylamide gel electrophoresis followed by quantitative dye-binding analysis shows that the molar ratio of the three components in neurofilaments isolated from bovine spinal cord myelinated nerve is 4:2:1 (NF3:NF2:NF1). Comparison of the molar ratios of each component in neurofilaments isolated from rat, bovine, and human brain shows a variation in the ratio of each of these polypeptides and raises questions about the physiological uniqueness of the molar composition of the neurofilament triplet. Reconstitution of the three bovine polypeptides into 10-nm filaments was accomplished under conditions in which the NF3 protein was limiting. Reassembly of 10-nm filaments with varying amounts of NF2 and NF1 indicate that the NF3 homopolymer has a limiting capacity to bind NF2 and NF1 and is saturated at a molar ratio of 2:2:1 (NF3:NF2:NF1). Isolation of the neurofilament complex at various stages of rat brain maturation indicates that NF3 and NF2 are integrated into the neurofilament complex as early as embryonic day 17, while NF1 copurifies with these proteins at postnatal day 16, eventually reaching a molar stoichiometry of 2:2:1 in the adult rat. The molecular stoichiometry of the neurofilament proteins, the differential integration of these proteins during brain development, and the variation of the molar composition between mammalian species suggest accessory roles for the NF2 and NF1 proteins in the neurofilament complex.     

Comparative studies of the neurofilament triplet protein peptide mapping by chemical cleavage

Peptide mapping of the three neurofilament protein subunits with apparent mol. weights of 210 kDa, 160 kDa and 70 kDa was performed with two different reagents: CNBr, BNPS-Skatole leading to the cleavage of methionyl and tryptophanyl bonds respectively. With BrCN we obtained two large fragments resistant to the cleavage, with mol. wts of 85 kDa for the 160 kDa and 135 kDa for the 210 kDa neurofilament proteins respectively. These fragments were located on the C-terminal part of the proteins (the tails) and correspond to specific regions responsible for their physiological identity. On the other hand, the cleavage with BNPS-Skatole at the tryptophanyl bonds gave similar patterns. The 210 kDa and 160 kDa neurofilament proteins gave a doublet of high mol. wt resistant to the cleavage, corresponding very likely to the C-terminal part and 4 fragments of mol. wt between 30 and 40 kDa corresponding to the N-terminal part. The neurofilament triplet share a common 30.5 kDa fragment located on the N-terminal part. From these peptide mapping studies, we conclude that the two neurofilament subunit proteins with mol. wts of 160 kDa and 210 kDa are different but related structures and that the CNBr characterized cleavage fragments of mol. wt 85,000 and 135 kDa are suitable polypeptides for sequence and immunological studies of the C-terminal part of these proteins.     

Third form of calcium-activated neutral proteinase from calf brain: purification, partial characterization and comparison of properties with other forms

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.     

Proteolysis of the neurofilament 68 kDa protein explains several previously described brain proteins of unique composition and high acidity

Neurofilaments follow the structural principles of non-neuronal intermediate filaments but contain additional sequences which are carboxyterminally located and increase in length between triplet proteins (68 kDa, 160 kDa and 200 kDa). The tailpiece domain has been sequenced in the case of the porcine 68 kDa protein. It has a unique amino acid composition. Within 106 residues there are only 12 different amino acid types, and glutamic acid accounts for 46% of the sequence. Examination of the literature on highly acidic brain proteins leads us to the proposal that microglutamic acid-rich protein, Glu-50, macroglutamic protein, as well as some unusual components of the S100 class, are most likely proteolytic degradation products of the neurofilament 68 kDa protein.     

Neurofilament Proteins in Cultured Chromaffin Cells

Antibodies were raised against the 200-kd, 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti-neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.     

Identification of Ca2+-Activated Neutral Protease in the Peripheral Nerve and Its Effects on Neurofilament Degeneration

Rat sciatic nerve segments were incubated in five different media. Disappearance of neurofilament (NF) triplet proteins (200K, 160K, and 68K MW) occurred in medium containing Ca²⁺ and was inhibited by the addition of E-64-c or leupeptin. Therefore, the presence in the peripheral nerve of an enzyme whose properties are similar to those of Ca²⁺-activated neutral protease (CANP) is suggested. The extraction of crude CANP from rat sciatic nerve was performed. CANP activity was completely recovered (0.129 ± 0.008 U/g) in the precipitate salted out by the addition of 0 to 50% saturated ammonium sulfate to the soluble fraction of the peripheral nerve (crude CANP). Properties of the crude CANP were examined using NF as a substrate and were found to be similar to those of the CANP extracted from skeletal muscle. Identification of the crude CANP with the CANP extracted from rat skeletal muscle was performed using the immunoreplica method. Bands corresponding to 73K were detected in both CANPs.     

Activation of intracellular calcium-activated neutral proteinase in erythrocytes and its inhibition by exogenously added inhibitors

Intracellular calcium-activated neutral proteinase (CANP) in rabbit erythrocytes was activated by an influx of Ca2+ into the cells. The catalytic large subunit changed from the original 79 kDa from to the 77 kDa and 76 kDa forms on activation just in the same manner as occurs in the autolytic activation of purified CANP in vitro. The activation required both extracellular Ca2+ and A23187, and was accompanied by the degradation of some membrane proteins and morphological changes in erythrocyte shape from discocytes to echinodisks, echinocytes, and spherocytes. Exogenously added Cbz-Leu-Leu-Leu-aldehyde inhibited the activation of intracellular CANP as well as the degradation of membrane proteins and the morphological changes indicating that the latter two processes are due to the action of CANP. Leupeptin and E64d were without effect on intracellular CANP.     

Properties of highly viscous gels formed by neurofilaments in vitro. A possible consequence of a specific inter-filament cross-bridging.

Neurofilaments freshly isolated from bovine spinal cord form a reversible gel in vitro, consisting of nearly parallel and interlinked filaments organized in bundles. This phenomenon is obtained above a critical neurofilament concentration and is highly sensitive to denaturation. No gelation occurs with neurofilaments reconstituted from urea-solubilized subunits. The velocity of the gelation kinetics, optimum at a slightly acidic pH, is inhibited by low and high ionic strength and activated by millimolar concentrations of Mg2+ and other bivalent cations. No protein other than the purified neurofilament preparation itself (80-95% neurofilament triplet) is necessary for the formation of a gel. However, purified cytoskeletal proteins from microtubules and neurofilaments influence the viscosity of the native preparation. These observations suggest a reticulation in vitro between neurofilaments, dependent upon a fragile conformation of the polymers and possibly mediated through the high-Mr neurofilament subunits (200 kDa and 150 kDa). The significance of these results is discussed with regard to the inter-neurofilament cross-bridging in situ involving the 200 kDa subunit described by Hirokawa, Glicksman & Willard [(1984) J. Cell Biol. 98, 1523-1536].     

An isoelectric variant of the 150,000-dalton neurofilament polypeptide. Evidence that phosphorylation state affects its association with the filament

A soluble isoelectric variant of the 150,000-dalton neurofilament protein was isolated from bovine brain by treating a partially purified filament preparation with a low-ionic-strength high-pH buffer. The protein (S150) had similar peptide maps to the neurofilament component of the same molecular weight (NF150) and was recognized by a polyclonal antibody made against the NF150 polypeptide. However, only half the anti-NF150 activity could be removed with the S150 protein. In addition, the S150 protein had a higher isoelectric point than the NF150 protein. Phosphate analysis indicated that the S150 protein was considerably lessened in phosphate content, which could account for the higher isoelectric point of the protein. It appears, therefore, that the S150 protein may be a precursor of NF150 or the result of phosphatase activity during the isolation procedure. Assembly studies showed that the S150 protein, unlike the NF150 protein, could not assemble with the 70-kDa neurofilament protein, indicating that the phosphate groups which were removed are important in the association of this protein to the neurofilament. When filaments containing all three triplet neurofilament polypeptides or those composed of the 70- and 150-kDa neurofilament proteins were subjected to acid phosphatase, a soluble fraction was obtained, which contained isoelectric variants with higher pI values than the NF150 polypeptide. Only unmodified NF150 protein was found in the insoluble fraction. These results support the argument that removal of phosphate groups results in the dissociation of this protein from the filament.     

Characterization of NADH-dependent Fe3+-chelate reductases of maize roots

Iron-deficient maize seedlings exhibit a starvation syndromecharacterized by an increase in different parameters such asroot fresh weight (+ 30%), protein (+ 25%) and plasma membrane-associatedNADH Fe³⁺ –EDTA reductase (NFR; +45%). NFR activity wasfound associated with 9 000g (20 min) and 110 000 g (1 h) sediments,purified plasma membrane and 110000 g supernatants. No differenceswere observed between the properties of reductases from Fe-starvedversus Fe-sufficient roots. The characterization of NFR wasundertaken. Low Mr forms (46 and 28 kDa, as detected by size-exclusionchromatography) were present in all fractions whereas 210 and110 kDa forms were unique in membranes and 110 000 g supernatants,respectively. The 210 kDa form was solubilized from microsomes and characterized.The enzyme is cetone-resistant and appears to be comprised largelyif not totally of the low Mr forms (46 and 28 kDa, correspondingto 30 and 32 kDa bands, respectively, in SDS-PAGE). The 210kDa form tended to break down to subunits following dilution,and the effect could be prevented by addition of 10% (v/v) glycerol.A three-step purification procedure for microsomal NFR was devised,consisting of acetone fractionation of lysophosphatidycholinesolubilized microsomes, Blue Sepharose CL-6B affinity chromatographyand a final size exclusion chromatography in the absence ofdetergent, resulting in a 700-fold purification of the 28 kDaprotein. The best electron acceptor for the purified 28 kDaform was ferricyanide (400µmol min^–1mg^–1 protein)followed by Fe³⁺–chelates (up to 200µol min^–1mg^–1 protein) and other compounds to a lesser extent (cytc, DCPIP).The 46 kDa form, on the other hand, had high ferricyanidereductase activity (about 300µmol min^–1mg^–1protein) and relatively low Fe³⁺–chelate reductase activity.The properties of NFR (high M, active forms, donor and acceptorspecificity, purification behaviour, large hydrophilic domains,size of subunits) suggest a relationship with the NADH-cyt b5reductase family of FAD-containing proteins. None of the latterflavoproteins is a transmembrane enzyme. Key words: Maize roots, Fe³⁺–reductase, ferricyanide reductase, iron     

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.     

A calcium-dependent protease associated with the neural cytoskeleton. Purification and partial characterisation

Calcium-dependent protease activity was found associated with a neurofilament-enriched cytoskeleton isolated from the bovine spinal cord. The protease was extracted from the cytoskeleton by 0.6 M KCl, and purified to apparent homogeneity (3300-fold) by chromatography on organomercurial-Sepharose 4B, casein-Sepharose 4B, and Sepharose CL-6B. A cytosolic calcium-dependent protease was similarly purified from the bovine spinal cord, after the cytosol was fractionated on DEAE-cellulose. Both cytoskeleton-bound and cytosolic enzymes had an apparent molecular mass of 100 kDa as judged by gel filtration, and consisted of two subunits (79 kDa and 20 kDa) upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both enzymes exhibited caseinolytic activity with 0.5 mM Ca2+ and above, and the activity was strongly inhibited by various thiol protease inhibitors. In the presence of 0.1-0.2 mM Ca2+, the 68-kDa and 160-kDa components, and to a lesser extent the 200-kDa component, of the neurofilament triplet polypeptides were degraded by the cytosolic protease, whereas the cytoskeleton-bound protease needed two-fold higher concentration of Ca2+ to degrade the neurofilaments. Nevertheless, the cytoskeleton-bound protease in situ, i.e. before its extraction form the cytoskeleton by 0.6 M KCl, preferentially degraded the 160-kDa component in the presence of 0.1-0.2 mM Ca2+, suggesting that a proper locational relation of this enzyme to the neurofilament structure is a prerequisite to its preference for the 160-kDa component. It appears that a factor or factors involved in such an interaction between the protease and the neurofilament were eliminated during the course of enzyme purification. The glial fibrillary acidic protein was almost insensitive to the proteases purified in the present study.     

Study of the 10-nm-filament fraction isolated during the standard microtubule preparation.

The cold non-depolymerizable fractions obtained during the standard procedure for the isolation of microtubules from ox brain stem-cerebral hemispheres and spinal cord have been studied. The cerebral-hemisphere preparation was composed of 10-nm filaments but also contained large amounts of membranes. The polypeptide content included tubulin, microtubule-associated proteins and minor proteins corresponding to the neurofilament triplet of proteins of mol.wt. 210 000, 160 000 and 70 000 respectively. The brain-stem preparation contained more 10-nm filaments than membranes. The polypeptide content consisted of the neurofilament triplet (35%), tubulin (30%) and minor proteins. In contrast, the spinal-cord preparation was mainly composed of 10-nm filaments, free of membranes and containing essentially the neurofilament protein triplet (64%). These filaments appeared very similar to the peripheral-nervous-system neurofilaments described by several authors. Since the best neurofilament from the central nervous system often contained less than 15% of the neurofilament protein triplet, our spinal-cord preparation is an improvement on the usual neurofilament preparation. This simple and rapid method gave large amounts of 10-nm filaments (100 mg per 100 g of spinal cord) characterized by the absence of membranous material, a low content of tubulin and the 50 000-mol.wt.-protein component, and a high content of neurofilament peptides. Thus, the presence of tubulin in 10-nm filament preparations seems to be related to the contaminant membranous material and not to be linked to the interaction in vitro of tubulin or microtubules with neurofilaments, as has been suggested previously.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Calcium-Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms

Abstract Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa²⁺ for half-maximal activation and 2 mM Ca²⁺ for optimal activity toward [methl-¹⁴C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.