Paola Strocchi

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 comparison of neurofilament proteins from rat and mouse cns.

Rat and mouse CNS neurofilament proteins (NFPs) were characterized and compared, in terms of electrophoretic properties on polyacrylamide gels and by peptide mapping, with one another and with other co‐purifying lower‐molecular‐weight CNS proteins, including α and β tubulin. NFPs were partially purified by modification of the axon flotation procedure of Norton and co‐workers and were demyelinated with Triton X‐100. On one‐dimensional SDS polyacrylamide gels the molecular weights of the triad of NFPs from both rat and mouse were approximately 200,000, 140,000, and 70,000. Prominent lower‐molecular‐weight proteins (63,000‐16,000) as well as minor amounts of tubulin and actin were observed after gel electrophoresis. On two‐dimensional gels (isoelectric focusing followed by SDS gel electrophoresis) each of the NFPs appeared to be composed of more than one component and the corresponding NFPs from rat and mouse had similar isoelectric points. Gel electrophoresis peptide mapping using Staphylococcus aureus V8 protease indicated the following: (1) the triad of NFPs of different sizes have different peptide maps; (2) α and β tubulin have nonidentical digestion products, which are dissimilar to those of the NFPs; (3) other proteins that co‐purify by the axon flotation procedure also have nonidentical peptide maps; and (4) the corresponding NFPs from rat and mouse have similar peptide maps. The co‐purifying proteins examined in detail (63,000–49,000) do not appear to be derived by proteolytic cleavage of NFPs and may represent other cytoskeletal constituents.

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.

The preparation of biologically active messenger RNA from human postmortem brain tissue.

Abstract: Messenger RNA (mRNA) was extracted from human postmortem brain tissue by alkaline phenol extraction of polysomes followed by oligo (dT)‐cellulose chromatography. The mRNA preparations stimulated protein synthesis in a cell‐free system containing wheat germ homogenate. The products of protein synthesis were analyzed by one‐ and two‐dimensional gel electrophoresis. These analyses indicated that numerous polypeptides, including tubulin subunits and actin isomers, were synthesized by the human mRNA. The molecular weight range of polypeptides synthesized by human mRNA fractions from two brain specimens were identical, and analysis by two‐dimensional gel electrophoresis indicated qualitatively similar products. The yield of mRNA extracted per gram of human tissue was less than the yield obtained with rat forebrains from animals sacrificed immediately before brain removal and mRNA purification. A decrease in the amount of polysomes isolated from human tissue relative to rat brain tissue was a major factor contributing to the low yield. The molecular weight distribution of polypeptides synthesized by human and rat brain mRNA fractions in wheat germ homogenate was similar; thus, there was no indication for selective breakdown or inactivation of high molecular weight mRNA species in the human tissue. Our studies indicate that it is possible to utilize postmortem tissue for molecular biological investigations of human brain mRNA.

In Vitro Synthesis of Human Brain Proteins Including Tubulin and Actin by Purified Postmortem Polysomes

Abstract: Polysomes were prepared from human brain tissue 2‐6 h postmortem; the polysomes were active in a cell‐free protein synthesis system containing rabbit reticulocyte factors. Protein synthesis was totally dependent upon added MgCl2, ATP, the reticulocyte factor fraction, and the human polysome fraction. Human brain proteins synthesized in the presence of L‐[35S]methionine were analyzed by one‐ and two‐dimensional polyacrylamide gel electrophoresis. Over 250 proteins were synthesized and they extended in size up to 250,000 d; many of the most abundant native human brain proteins were synthesized, including tubulin and actin. It was shown that human brain α and β tubulin and actin isomers synthesized in vitro from human postmortem polysomes have the same apparent molecular weights and isoelectric points as the corresponding proteins synthesized by rat polysomes from fresh cortices. The corresponding tubulin and actin synthesized by human and rat brain polysomes also yield the same radioactive methionine‐containing peptides after digestion with Staphylococcus aureus V8 protease. These analyses indicate that postmortem polysomes contain active messenger RNA which can direct the partial and/or complete synthesis of actin and tubulin subunits and other human brain proteins.

MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two‐dimensional gels and one‐dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S1 and S2, with similar DNase I affinity were also isolated. S11 and S2 have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S1 and S2 is 3‐fOld greater in synaptosomes when compared to similar non‐synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I‐actin complex.

BIOSYNTHESIS OF HETEROGENEOUS FORMS OF MAMMALIAN BRAIN TUBULIN SUBUNITS BY MULTIPLE MESSENGER RNAs

—Heterogeneity among the primary translation products of rat brain tubulin messenger RNA was examined. On two‐dimensional gels native cytoplasmic tubulin from randomly bred rats (PB21) consists of two groups of α tubulin subunits among which the most acidic forms, α1 and α2, are most abundant; and β tubulin consists of a minimum of two species, β1 and β2. In the same group of animals the primary translation products of rat brain tubulin mRNA consist of at least these four subunit forms (α1α2, β1 and β2); however, minor basic forms of α subunits were not synthesized. This same result was obtained from a homologous brain protein synthesizing system, a heterologous system prepared from brain polysomes and rabbit reticulocyte initiation factors, and a wheat germ lysate programmed with brain poly A mRNA. A variant form of brain tubulin was found in rats bred monogamously for over 30 generations (MB71 rats). MB71 brain polysomes synthesize overlapping a subunits which migrate in two‐dimensional gels to the α1 position, and the typical PB21 α2 is not present. The addition of PB21 brain mRNA to a protein synthesizing system composed of MB71 polysomes plus reticulocyte initiation factors allowed synthesis of the typical α2 tubulin in addition to the MB71 tubulin subunits. The structural relationship among subunits was examined by radioiodinated peptide mapping. The α subunits are structurally different from the β subunits; however, among the major tyrosine‐containing tryptic peptides no prominent differences were observed between α1 and α2, or between β1 and β2 by the radioiodination procedure. The results provide evidence for heterogeneity among the primary translation products of brain tubulin mRNA, and for the existence of multiple functional tubulin genes in rat brain.

Studies on the biosynthesis of intermediate filament proteins in the rat CNS.

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell‐free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two‐dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.

Subunit structure of synaptosomal tubulin

The subunit structure of rat brain synaptosomal tubulin was examined by high resolution two-dimensional gel fractionation. Whole brain cytoplasmic tubulin consists of two groups of alpha subunits (alpha1 and alpha2), and a minimum of two beta subunits (beta1 and beta2). Both alpha subunits consist of a major relatively acidic form and minor relatively basic forms. In contrast, tubulin purified from synaptoplasm contains an additional subunit, alpha3, which has the same isoelectric point but slightly faster electrophoretic mobility than alpha1 and alpha2. All synaptosomal alpha subunits are the relatively acidic forms and the minor basic forms are absent. The synaptosomal beta subunits have electrophoretic properties similar to the corresponding cytoplasmic forms. The alpha3 synaptosomal tubulin subunit has affinity for colchicine, has a tryptic peptide map similar to whole brain cytoplasmic alpha tubulin, and can be purified by a standard tubulin purification method.

Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane‐Bound Polysomes

Abstract: Free and membrane‐bound polysomes were prepared from rat forebrain and added to a cell‐free system containing rabbit reticulocyte factors and L‐[35S]methionine. The translation products were analyzed by two‐dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α1, α2, β1, and α2) that are found in rat forebrain cytoplasm. The membrane‐bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two‐dimensional gel. MB has a molecular weight and isoelectric point similar to α‐tubulin. Only trace amounts of α‐ and β‐tubulin and actin were synthesized by the membrane‐bound polysomes. MB co‐purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane‐bound polysomes) and α‐ and β‐tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α‐ and β‐tubulin; MB yielded peptides not found in tubulin. We conclude that membrane‐bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane‐bound polysomes and is similar, but not identical, to α‐tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.