Purification of calcium-activated neutral proteinase (CANP) from purified myelin of bovine brain white matter

A calcium-activated neutral proteinase was purified from myelin of bovine brain white matter. Myelin purified in the presence of EDTA (2 mM) was homogenized in 50 mM Trisacetate buffer at pH 7.5, containing 4 mM EDTA, 1 mM NaN₃, 5 mM -mercaptoethanol and 0.1% Triton X-100 for two hours. After centrifugation at 87,000g for 1 hour, the supernatant was subjected to purification through successive column chromatography as follows: i) DEAE-cellulose, ii) Ultrogel (AC-34) filtration, iii) Phenyl-Sepharose, iv) a second DEAE-cellulose. The enzyme activity was assayed using azocasein as substrate. The myelin enzyme was purified 2072-fold and SDS-PAGE analysis of the purified enzyme revealed a major subunit of 72–76 K. The enzyme was inhibited by iodoacetate (1 mM), leupeptin (1 mM), E-64C (1.6 mM), EGTA (1 mM), antipain (2 mM) and endogenous inhibitor calpastatin (2 g). It required 0.8 mM Ca²⁺ for half-maximal activation and 5 mM Ca²⁺ for optimal activation. Mg²⁺ (5 mM) was ineffective while Zn²⁺ and Hg²⁺ were inhibitory. The pH optimum was ranged from 7.5–8.5. Treatment of myelin with Triton X-100 increased the enzyme activity by 10-fold suggesting it is membrane bound whereas the purufied enzyme was not activated by Triton X-100 treatment. The presence of CANP in myelin may mediate the turnover of myelin proteins and myelin breakdown in degenerative brain diseases.     

Distribution of calcium activated neutral proteinase (mM CANP) in myelin and cytosolic fractions in bovine brain white matter

The activity of calcium-activated neutral proteinase (mM CANP) was determined in homogenate, myelin and supernatant of bovine brain corpus callosum. The enzyme activity in homogenate and myelin was increased eleven and thirteen-fold respectively by Triton X-100. Myelin prepared by the method of Norton and Poduslo as well as by a modified method, was shown to contain most (more than 50%) of homogenate mM CANP activity. The specific activity was highest in myelin, and increased almost three times more than the homogenate. Supernatant only contained 17% of enzyme activity. It is concluded from these studies that mM CANP is tightly bound to the membrane and predominantly associated with the myelin sheath.     

The presence of ATP + ubiquitin-dependent proteinase and multicatalytic proteinase complex in bovine brain

The presence of two distinct high-molecular-weight proteases with similar pH optima in the weakly alkaline region was shown in cytosol of the bovine brain cortex. They were separated by ammonium sulfate fractionation and each was further purified by DEAE-Sephacel Sephacryl S-300, DEAE-Cibacron Blue 3GA-agarose, heparin-agarose, and Sepharose 6B chromatography. The larger enzyme (Mr 1,400 kDa), which precipitates at 0–38% ammonium sulfate saturation, seems to be active in ATP+ubiquitin (Ub)-dependent proteolysis; it has low basal caseinolytic activity that is stimulated 3-fold by ATP, and when Ub is present ATP causes a 4.5-fold stimulation. A second proteinase was also found to be present (Mr 700 kDa) that precipitates at 38–80% ammonium sulfate saturation, is composed of multiple subunits ranging in Mr from 18 to 30 kDa, and degrades both protein and peptide substrates, demonstrating trypsin-, chymotrypsin- and cucumisin-like activities. Catalytic, biochemical, and immunological characteristics of this proteinase indicate that it is a multicatalytic proteinase complex (MPC), whose enzyme activity, in contrast to that of MPC from bovine pituitaries (1–3), is stimulated 1.7-fold by addition of ATP in the absence of ubiquitin at the early steps of purification; this property is lost during the course of further purification. Both proteinases are present in the nerve cells, since the primary chicken embryonic telencephalon neuronal cell culture extracts contain both ATP+Ub-dependent proteinase and MPC activities.Special issue dedicated to Dr. Paola S Timiras     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Triton X-100 extractions of central nervous system myelin indicate a possible role for the minor myelin proteins in the stability of lamellae

Isolated CNS myelin membranes were extracted with Triton X-100 under conditions previously established for the isolation of cytoskeletal proteins. Treated myelin retained much of its characteristic lamellar structure despite the removal of most of the major myelin basic protein (18.5 kDa) and the proteolipid protein, which together normally constitute 60% of the total myelin protein. The SDS-PAGE profile of this extract residue demonstrated an enrichment in proteins of Mr 30 to 60 kilodaltons (the Wolfgram group). The major myelin proteins were identified by antibodies on Western immunoblots, as were the 23-cyclic nucleotide 3-phosphodiesterase (CNP), actin, tubulin, myelin-associated glycoprotein (MGP) and the 21.5 kDa MBP. The overall behavior of CNP, the 21.5 kDa MBP, MGP and tubulin towards Triton extraction is reminiscent of the behavior of other membrane-skeletal complexes, supporting the idea that these and other minor myelin proteins might be part of heteromolecular complexes with interactions spanning several lamellae of the myelin sheath.     

Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

Quantitative distribution of calcitonin gene-related peptide in the rat central nervous system

Using an antiserum directed against human calcitonin gene-related peptide (hCGRP), which fully cross reacts with rat CGRP, a sensitive radioimmunoassay was developed. The antiserum was characterized by displacement curve characteristics and high performance liquid chromatography. The assay was applied to rat brain tissue and the concentration of CGRP for 48 microdissected brain areas is presented. Highest levels (1000–4500 fmol/mg protein) were found in the central amygdaloid, caudate putamen, and spinal trigeminal nerve nucleus and tract, substantia gelatinosa, and the dorsal horn of the spinal cord. Moderate levels (200–600 fmol/mg protein) were found in the bed nucleus of the stria terminalis, the subfornical organ, the paraventricular, arcuate, dorsomedial, dorsal parabrachial, ambiguus and tractus solitarii nuclei and in the median eminence. These results coincide with those previously obtained by immunohistochemistry. The widespread distribution in the brain suggests involvement of CGRP in a variety of behavioral functions.     

Microstructural organization of the central nervous system in the harvestman Leiobunum japonicum (Arachnida: Opiliones)

Although the order Opiliones constitutes the third‐largest group of arachnids, this creature is still mysterious and has a rich unexplored field compared to what is known about insects and crustaceans. The order Opiliones is traditionally regarded as a close relative of mites, mainly because of morphological similarities in external body structure; however microstructural organization of the ganglionic neurons and nerves in the harvestman Leiobunum japonicum is quite similar to the central nervous system (CNS) in all extant arachnids. The CNS consists of a large neural cluster with paired appendicular nerves. The esophagus passes through the neural cluster and divides it into the upper supraesophageal ganglion (SpG) and the lower subesophageal ganglion (SbG). The dorsal part of the SpG has a quite condensed cell body compared with other parts of the CNS and has two main components, the protocerebrum and the cheliceral ganglion. The protocerebrum receives the optic nerves and has four main groups of neuropiles from the optic lobes, the superior central body, the lateral neuropils (corpora pedunculata) and the inferior neuropil. However, a pair of pedipalpal and four pairs of appendage nerves including several pairs of abdominal nerves arise from the nerve masses of the SbG.     

Autoradiographic distribution of 125I calcitonin gene-related peptide binding sites in the rat central nervous system

Using autoradiographic method and 125I-Tyro rat CGRP as a ligand, receptor binding sites were demonstrated in the rat central nervous system. Saturation studies and Scatchard analysis of CGRP-binding to slide mounted tissue sections containing primarily cerebellum showed a single class of receptors with a dissociation constant of 0.96 nM and a Bmax of 76.4 fmol/mg protein. 125I-Tyro rat CGRP binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the telencephalon (medial prefrontal, insular and outer layers of the temporal cortex, nucleus accumbens, fundus striatum, central and inferior lateral amygdaloid nuclei, most caudal caudate putamen, organum vasculosum laminae terminalis, subfornical organ), the diencephalon (anterior hypothalamic, suprachiasmatic, arcuate, paraventricular, dorsomedial, periventricular, reuniens, rhomboid, lateral thalamic pretectalis and habenula nuclei, zona incerta), in the mesencephalon (superficial layers of the superior colliculus, central nucleus of the geniculate body, inferior colliculus, nucleus of the fifth nerve, locus coeruleus, nucleus of the mesencephalic tract, the dorsal tegmental nucleus, superior olive), in the molecular layer of the cerebellum, in the medulla oblongata (inferior olive, nucleus tractus solitarii, nucleus commissuralis, nuclei of the tenth and twelfth nerves, the prepositus hypoglossal and the gracilis nuclei, dorsomedial part of the spinal trigeminal tract), in the dorsal gray matter of the spinal cord (laminae I-VI) and the confines of the central canal. Moderate receptor densities were found in the septal area, the "head" of the anterior caudate nucleus, medial amygdaloid and bed nucleus of the stria terminalis, the pyramidal layers of the hippocampus and dentate gyri, medial preoptic area, ventromedial nucleus, lateral hypothalamic and ventrolateral thalamic area, central gray, reticular part of the substantia nigra, parvocellular reticular nucleus. Purkinje cell layer of the cerebellum, nucleus of the spinal trigeminal tract and gracile fasciculus of the spinal cord. The discrete distribution of CGRP-like binding sites in a variety of sensory systems of the brain and spinal cord as well as in thalamic and hypothalamic areas suggests a widespread involvement of CGRP in a variety of brain functions.     

Cytosolic calcium dependent neutral proteinase of human erythrocytes: the role of calcium ions on the molecular and catalytic properties of the enzyme

The soluble neutral proteinase of human erythrocytes dissociates into constituent subunits of 80k and 30k in the presence of mM concentrations of Ca²⁺. Similarly the soluble natural inhibitor of this proteinase, of approximate molecular weight 240k, is dissociated into 60k subunits by mM concentrations of Ca²⁺. Removal of Ca²⁺ restores the native oligomeric structure of the proteinase and of the natural inhibitor. The formation of the native active enzyme or of the inactive enzyme-inhibitor complex depends on reversible association-dissociation processes mediated by Ca²⁺ concentration.     

UDP-N-acetylglucosaminyl transferase (OGT) in brain tissue: temperature sensitivity and subcellular distribution of cytosolic and nuclear enzyme

In brain tissue, UDP-N-acetylglucosaminyl transferase (OGT) is known to catalyze the addition of a single N-acetylglucosamine moiety (GlcNAc) onto two proteins linked to the etiology of neurodegenerative disease--beta-amyloid associated protein and tau. Hyperphosphorylation of tau appears to cause neurofibrillary tangles and cell death, and a functional relationship appears to exist between phosphorylation and glycosylation. Since a greater understanding of brain OGT may provide new insights into the pathogenesis of Alzheimer's disease, we examined the characteristics and subcellular distribution of OGT protein and OGT activity and its relationship to O-linked glycosylation. We found that cytosolic OGT activity is 10 times more abundant in brain tissue compared with muscle, adipose, heart, and liver tissue. Temperature studies demonstrated that cytosolic OGT activity was stable at 24 degrees C but was rapidly inactivated at 37 degrees C (T1/2 = 20 min). Proteases were probably not involved because OGT immunopurified from cytosol retained temperature sensitivity. Subcellular distribution studies showed abundant OGT protein in the nucleus that was enzymatically active. Nuclear OGT activity exhibited a high affinity for UDP-GlcNAc and a salt sensitivity that was similar to cytosolic OGT; however, nuclear OGT was not inactivated at 37 degrees C, as was the cytosolic enzyme. Two methods were used to measure O-linked glycoproteins in brain cytosol and nucleosol -[3H]galactose labeling and western blotting using antibodies against O-linked glycoproteins. Both methods revealed a greater abundance of O-linked glycoproteins in the nucleus compared to cytosol.     

Activity and subcellular distribution of protein kinase C (PKC) in muscle and brain of force-fed zinc-deficient rats

The purpose of the present study was to investigate, in force-fed rats, whether alimentary zinc (Zn) deficiency affects the activity of the Zn-metalloenzyme protein kinase C (PKC). The in vivo activity of PKC was determined by measuring the subcellular distribution of the enzyme between the cytosolic and the particulate fraction in brain and muscle. For this purpose, 24 male Sprague-Dawley rats with an average live mass of 126 g were divided into 2 groups of 12 animals each. The Zn-deficient and the control rats received a semisynthetic casein diet with a Zn content of 1.2 and 24.1 ppm, respectively. All animals were fed four times daily by gastric tube in order to ensure that the depleted animals also received adequate nutrients and to synchronize the feed intake exactly. After 12 d, the depleted rats were in a state of severe Zn deficiency, as demonstrated by a 70% lower serum Zn concentration and a 66% reduction in the serum activity of alkaline phosphatase. Neither the cytosolic nor the particulate fraction of the thigh muscle showed any difference between the depleted and the control animals as regards PKC activity/g of muscle. The specific activity of PKC/mg of protein in the cytosolic fraction of the muscle was not affected by alimentary zinc deficiency, whereas the specific activity of PKC in the particulate fraction of the muscle was reduced by a significant 10% in Zn deficiency (150±12 vs 135±14 pmol P/min/mg protein). In the brain, neither the cytosolic nor the particulate fraction revealed any difference in PKC activity/g of fresh weight or in the specific activity/mg of protein between the control and the Zn-deficient rats.     

Molecular characterization of the detergent-insoluble cholesterol-rich membrane microdomain (raft) of the central nervous system

Many fundamental neurological issues such as neuronal polarity, the formation and remodeling of synapses, synaptic transmission, and the pathogenesis of the neuronal cell death are closely related to the membrane dynamics. The elucidation of functional roles of a detergent-insoluble cholesterol-rich domain (raft) could therefore provide good clues to the molecular understanding of these important phenomena, for the participation of the raft in the fundamental cell functions, such as signal transduction and selective transport of lipids and proteins, has been elucidated in nonneural cells. Interestingly, the brain is rich in raft and the brain-derived raft differs in its lipid and protein components from other tissue-derived rafts. Since many excellent reviews are written on the membrane lipid dynamics of this microdomain, signal transduction, and neuronal glycolipids, we review on the characterization of the raft proteins recovered in the detergent-insoluble low-density fraction from rat brain. Special focus is addressed on the biochemical characterization of a neuronal enriched protein, NAP-22, for the lipid organizing activity of this protein has become increasingly clear.     

Physiological and pathological roles of interleukin-6 in the central nervous system

The cytokine interleukin-6 (IL-6) is an important mediator of inflammatory and immune responses in the periphery. IL-6 is produced in the periphery and acts systemically to induce growth and differentiation of cells in the immune and hematopoietic systems and to induce and coordinate the different elements of the acute-phase response. In addition to these peripheral actions, recent studies indicate that IL-6 is also produced within the central nervous system (CNS) and may play an important role in a variety of CNS functions such as cell-to-cell signaling, coordination of neuroimmune responses, protection of neurons from insult, as well as neuronal differentiation, growth, and survival. IL-6 may also contribute to the etiology of neuropathological disorders. Elevated levels of IL-6 in the CNS are found in several neurological disorders including AIDS dementia complex, Alzheimer's disease, multiple sclerosis, systemic lupus erythematosus, CNS trauma, and viral and bacterial meningitis. Moreover, several studies have shown that chronic overexpression of IL-6 in transgenic mice can lead to significant neuroanatomical and neurophysiological changes in the CNS similar to that commonly observed in various neurological diseases. Thus, it appears that IL-6 may play a role in both physiological and pathophysiological processes in the CNS.     

Immunoassay and Activity of Calcium-Activated Neutral Proteinase (mCANP): Distribution in Soluble and Membrane-Associated Fractions in Human and Mouse Brain

The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.     

Cellular and subcellular distribution of the calcium-binding protein NCS-1 in the central nervous system of the rat

NCS-1 (neuronal calcium sensor) is a recently characterized member of a highly conserved neuron-specific family of calcium-binding proteins, which also includes frequenin and recoverin. The cellular and subcellular distributions of NCS-1 in the rat nervous system were investigated using light- and electron-microscopic immunohistochemistry. NCS-1 immunoreactivity was localized to neuronal cell bodies and axons throughout the brain and spinal cord but not to glial cells. The most intense labeling was observed in myelinated axons, the axonal ramifications of the basket cell in the cerebellar cortex, and large neurons in the brainstem and pons. These same structures were also characterized by heavy labeling for neurofilament protein, as determined by double-labeling experiments. Most axon terminals were unlabeled or only lightly labeled. The most remarkable subcellular staining occurred in the perikarya where intense labeling was associated with the membranes of the trans saccules of the Golgi apparatus. The widespread distribution of NCS-1 indicates that it may be active in a variety of calcium-dependent neuronal functions, whereas the specific subcellular localization to the Golgi apparatus and neurofilament-rich structures suggests a specialized role in calcium regulated protein trafficking and cytoskeletal interactions.     

Development and sensitivity to serotonin of Drosophila serotonergic varicosities in the central nervous system

Serotonin is a classical small-molecule neurotransmitter with known effects on developmental processes. Previous studies have shown a developmental role for serotonin in the fly peripheral nervous system. In this study, we show that serotonin can modulate the development of serotonergic varicosities within the fly central nervous system. We have developed a system to examine the development of serotonergic varicosities in the larval CNS. We use this method to describe the normal serotonergic development in the A7 abdominal ganglion. From first to third instar larvae, the volume of the neuropil and number of serotonergic varicosities increase substantially while the varicosity density remains relatively constant. We hypothesize that serotonin is an autoregulator for serotonergic varicosity density. We tested the sensitivity of serotonergic varicosities to serotonin by adding neurotransmitter at various stages to isolated larval ventral nerve cords. Addition of excess exogenous serotonin decreases native varicosity density in older larvae, and these acute effects are reversible. The effects of serotonin appear to be selective for serotonergic varicosities, as dopaminergic and corazonergic varicosities remain qualitatively intact following serotonin application.     

Cellular and subcellular localization of 3H-diethylstilboestrol in the central nervous system

Summary The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 g of (monethyl-³H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for ³H-oestradiol-17 in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration of radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of ³H-oestradiol.This research was supported in part by US PHS Grant No. NS12933NIH Career Development Awardee No. NS00164The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.