Victor S. Sapirstein

Effect of retinoic acid on growth and morphological differentiation of mouse NB2a neuroblastoma cells in culture

We have characterized the effects of retinoic acid (RA) on the growth, morphology and biosynthesis of cytoskeletal proteins in NB2a mouse neuroblastoma cells. In addition, the morphological and biochemical changes were compared to those induced by dibutyryl cyclic AMP (db cAMP). Growth inhibition by RA was concentration-dependent and was first detected 24 h after addition of RA. The proliferation of RA-treated NB2a was more dependent on serum than was the proliferation of untreated cultures and RA decreased the saturation density of NB2a cells grown in serum. Morphological changes induced by RA include the formation of an elaborate network of branching neurites in NB2a cells. In contrast, neurites induced by db cAMP or serum deprivation were bipolar and unbranching. Ultrastructural observations of neurites induced by RA revealed dendritic characteristics such as polysomes, spines and absence of intermediate filaments, while neurites induced by db cAMP had axonal characteristics such as filament bundles, absence of ribosomes, and the formation of membrane densities when neurite endings contacted another cell body. These morphological differences were also reflected in a number of changes in the biosynthesis of cytoskeletal proteins. These results suggest that NB2a cells treated with RA and db cAMP are a model system for the study of distinct stages of differentiation.

Expression and distribution of microtubule-associated protein 2 (MAP2) in neuroblastoma and primary neuronal cells

We examined the expression and distribution of microtubule-associated protein 2 (MAP2) during the differentiation in culture of both mouse NB2a neuroblastoma and primary embryonic rat neurons. The differentiation of NB2a cells was induced with retinoic acid (RA) which stimulated the extension of a highly branched neuritic network and dibutyryl cAMP which stimulated the outgrowth of long bipolar or monopolar processes. We found that although monoclonal antibodies to MAP2 stained the cell bodies of control and differentiated cells, only the RA-induced neurites were positive for this antigen. These data support our ultrastructural studies indicating that the RA-induced neurites were dendrite-like and that the dibutyryl cAMP-induced processes were axon-like. Studies on the biosynthesis of MAP2 indicated that RA induced a 2-3-fold increase in MAP2 synthesis in 24 h; however, this effect was transient, with the synthesis of MAP2 in RA-treated cells returning to control level by 72 h. Although biosynthetic studies suggested the synthesis of species at 250-300 kdalton, the major molecular weight form in the neuroblastoma cells was 230 kdalton. Immunocytochemical analysis of primary neurons showed staining of neuronal cell bodies and of short processes, but virtually no staining of the long axon-like processes. The staining of neuronal cell bodies and processes was evident at all stages of cell differentiation. This finding was corroborated by immunoblots which showed significant amounts of MAP2 throughout cell development. The molecular weight of the immunoreactive material was ca. 300 kdalton in both primary neurons and rat brain. Immunoblots also revealed that embryonic neurons expressed only MAP2B as they differentiated in culture for 14 days. Biosynthesis studies suggested that early in culture there was a modest increase in MAP2 synthesis, but no detectable change was observed thereafter. We concluded therefore that both neuroblastoma cells and primary neurons can differentiate neuritic processes, which show dendritic properties in terms of morphology and preferential distribution of MAP2.

Phorbol ester stimulates the phosphorylation of rabbit erythrocyte band 4.1

The effect of phorbol esters on membrane phosphorylation was examined in intact rabbit erythrocytes prelabeled with [32P]orthophosphate. Tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate, but not the inactive 4 alpha-phorbol 12,13-didecanoate, specifically stimulated the phosphorylation of two proteins in the erythrocyte membrane. They have apparent molecular weight of 100,000 dalton and 85,000 dalton. When intracellular calcium concentrations were raised by ionomycin, A23187, both the 85,000 dalton polypeptide and the 85K phosphoprotein were degraded. The 85,000 dalton phosphoprotein showed cross-reactivity with antiserum to human erythrocyte band 4.1. Based on its susceptibility to calcium-activated protease and its immunological property, the 85,000 dalton phosphoprotein was identified to be the band 4.1 of the erythrocyte membrane skeletal network.

Properties and Function of Brain Carbonic Anhydrase

This chapter has described the characterization and biogenesis of soluble and membrane-bound CA in the central nervous system. The two forms of the enzyme appear to be quite similar in their molecular characteristics, however the data strongly indicate that they are synthesized on separate polysomal populations; the membrane-bound form resulting from synthesis on the RER. Our preliminary data suggest that the partitioning of mRNA for CA on the different polysomes results from the interaction of partial nascent chains with a specific receptor on the RER. We feel a function of membrane-associated synthesis is for the targeting of CA to sites in the cell where there are enzymes that can rapidly utilize the protons and bicarbonate produced by CA catalytic activity for ion exchange reactions. We have also presented arguments that CA may function as a bicarbonate source in the control of metabolism specifically in the acceleration of fatty acid synthesis in the oligodendrocyte.

Characterization and Biosynthesis of Soluble and Membrane-Bound Carbonic Anhydrase in Brain

Abstract: Carbonic anhydrase from both the cytoplasmic and membrane fractions of the forebrains of rats was characterized with respect to enzymatic activity, immunoreactivity, and in vitro biosynthesis. A procedure for the rapid purification of both membrane‐bound and soluble brain carbonic anhydrase is presented that permits retention of full enzymatic activity. Both forms of the enzyme were found to show specific activities of approximately 5500 Units/mg protein when CO2 hydrating activity was determined. In addition, they exhibited similar esterase activity when assayed with p‐nitrophenyl acetate. The membrane‐bound form, although requiring detergent for extraction from membranes, was freely soluble in aqueous buffers after purification. The molecular weights of both soluble and membrane‐bound carbonic anhydrase are 30,000 daltons, and mixing experiments failed to show any significant differences with respect to size. The two forms also exhibit isoelectric points of 7.2. However, the two proteins were found to differ in two respects. Complement fixation indicated that antibodies to soluble carbonic anhydrase had a higher affinity for the soluble form than for the membrane‐bound form. The failure to observe any precursor‐product relationship between these two proteins with pulse chase studies and the establishment that carbonic anhydrase‐like proteins are synthesized on both free polysomes and the rough endoplasmic reticulum indicated that these proteins are synthesized by two separate mechanisms. In vitro synthesis on both free and bound polysomes was determined by two independent methods using different antibodies and different analytical procedures. The basis for these findings and their physiologic importance are discussed.

Is Na + K ATPase a Myelin-Associated Enzyme?

Abstract: The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter‐enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. The discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29°C) than for the microsomal fraction (21°C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions. Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10−3 M‐strophanthidin. The evidence thus indicates that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggests that this is not a significant factor and that Na + K ATPase may be a myelin‐associated enzyme.

PURIFICATION OF MYELIN CARBONIC ANHYDRASE

Abstract— A procedure has been developed for the purification of the membrane bound form of carbonic anhydrase from rat brain myelin. The procedure is rapid, requiring only two steps, and can be applied to small amounts of material. Conditions have been established whereby the enzyme can be almost quantitatively solubilized with up to a 60 fold increase in specific activity. Purification by affinity chromatography yields a preparation which is homogeneous by polyacrylamide gel electrophoresis. However, preliminary evidence suggests that activity may be reduced by the removal of lipids during chromatography and subsequent dialysis. The purified preparation is high in dicarboxylic and hydroxyl amino acids and contains only 1×2 cysteine residues. The reduction of cysteine appears to be essential for the full expression of enzymatic activity.

Soluble and membrane bound carbonic anhydrases from rat CNS: regional development.

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.

Myelin-Associated Enzymes

For many years, myelin was viewed only as an inert membrane whose function was to act as an insulator around the nerve axon. Consequently, a major focus of myelin biochemical research was on the structural components of the membrane, and extensive studies were carried out on both the lipid and protein composition of different myelin membranes.1,2 Two proteins, myelin basic protein and proteolipid protein, constitute more than 85% of the proteins of CNS myelin, and emphasis was on the relatively simple protein composition of the membrane. There appeared to be no need to seek a function for the additional proteins that were present only in relatively small amounts and did not appear important for the maintenance of myelin structure.

Heterogeneity of microtubule-associated protein (MAP2) in vertebrate brains

We have utilized monoclonal antibodies to investigate the antigenic diversity of MAP2-immunoreactive proteins in the nervous system of vertebrates. We found that domains defined by the monoclonal antibodies differed in their conservation across vertebrate evolution, ranging from wide cross-reactivity with almost all vertebrates (mammals, birds, reptiles and amphibians) to a very limited cross-reactivity with only few mammalian species. However, we did not find MAP2-immunoreactive proteins in fish species with either of the monoclonal or polyclonal antibodies. There was also a significant divergence in the apparent molecular weight of MAP2, even in closely related species. For example, different species of wild mice and strains of laboratory mice showed variations of up to 30 kDa in their apparent molecular mass. Using alkaline phosphatase, under conditions that dephosphorylate neurofilaments, we showed that the observed heterogeneity was not the result of variations in the phosphate content. The heterogeneity in molecular weight of MAP2 may, therefore, be the result of changes in primary structure, transcriptional variations or different post-translational modifications. The heterogeneity of MAP2, as well as its specific distribution and implicated interactions with other molecules, underscore the complexity of MAP2 and its potential for structural and functional diversity. The phylogenic analysis of such a complex molecule also provides a method to establish the uniqueness of monoclonal antibodies and the degree of their conservation for their corresponding epitopes.