RAPID CONCURRENT AUTOMATED FLUORIMETRIC ASSAY OF NORADRENALINE, DOPAMINE, 3, 4-DIHYDROXYPHENYLACETIC ACID, HOMOVANILLIC ACID AND 3-METHOXYTYRAMINE IN MILLIGRAM AMOUNTS OF NERVOUS TISSUE AFTER ISOLATION ON SEPHADEX G10

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two-dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.     

On the Identification of α- and β-Tubulin Subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α-and β-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to α-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α₂ subunit of tubulin is tyrosylated.     

CHARACTERIZATION OF MULTIPLE FORMS OF BRAIN TUBULIN SUBUNITS

Abstract— Microtubular protein was isolated from rat forebrain by biochemical purification (ammonium sulfate precipitation followed by DEAE cellulose chromatography) or by two cycles of aggregation-disaggregation. The protein subunit structure was examined on two-dimensional electrophoretograms: first dimension, urea isoelectric focusing gel; second dimension, sodium dodecyl sulfate exponential acrylamide slab gel. Two forms of α tubulin were separated in the second dimension on the basis of different rates of migration (α and α₂). Each of these species was further separated into at least three forms with different isoelectric points. β Tubulin was separated into a minor species (BI) and a major species β₂). Multiple subunits were observed using protein from either purification method and in a two-dimensional electrophoretogram of total supernatant proteins from rat brain. Separation and visualization of multiple forms of α and β tubulin is consistent with reports that provide evidence for post-translational modification of these proteins.     

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.     

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.     

Allele frequencies of the major milk proteins in the Finnish Ayrshire and detection of a new K-casein variant

A total of 20990 Finnish Ayrshire cows were phenotyped for the major milk proteins by isoelectric focusing in polyacrylamide gels. The predominant alleles in the Finnish Ayrshire were α_S1-casein B (0.999), α_S2-casein A (0.991), β-casein A₁ (0.509) and α₂ (0.490), α₂₂-casein A (0.612) and β-lactoglobulin B (0.716). The K-casein E allele (0.307) was also rather common in the Finnish Ayrshire. A new K-casein variant (K-casein F) was demonstrated in two Finnish Ayrshire cows, a dam and a daughter.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

UV effects on photosynthesis and phycobiliprotein composition in the flagellate Cyanophora paradoxa

Abstract The effects of solar and artificial ultraviolet radiation on photosynthetic oxygen production and phycobiliprotein composition were investigated in the freshwater flagellate, Cyanophora paradoxa . The phycobiliproteins of the cell acting as photosynthetic accessory pigments were found to be readily affected by even short exposure to ultraviolet radiation, while the membrane-bound chlorophyll protein complexes were hardly impaired as demonstrated by SDS polyacrylamide gel electrophoresis, isoelectric focussing and fast protein liquid chromatography (FPLC). The phycobilisomes are dissembled from the outside inwards and go from higher molecular weight components to hexamers ( αβ )₆, trimers ( αβ )₃ and finally monomers (αβ). Fluorescence spectra indicated that the energy transfer from accessory pigments to the photosystems was impaired by ultraviolet radiation. Photosynthetic oxygen production was affected on a much faster timescale than changes in the absorption spectra or at the protein level.     

UV effects on photosynthesis and phycobiliprotein composition in the flagellate Cyanophora paradoxa

Abstract The effects of solar and artificial ultraviolet radiation on photosynthetic oxygen production and phycobiliprotein composition were investigated in the freshwater flagellate, Cyanophora paradoxa . The phycobiliproteins of the cell acting as photosynthetic accessory pigments were found to be readily affected by even short exposure to ultraviolet radiation, while the membrane-bound chlorophyll protein complexes were hardly impaired as demonstrated by SDS polyacrylamide gel electrophoresis, isoelectric focussing and fast protein liquid chromatography (FPLC). The phycobilisomes are dissembled from the outside inwards and go from higher molecular weight components to hexamers (αβ)₆, trimers (αβ)₃ and finally monomers (αβ). Fluorescence spectra indicated that the energy transfer from accessory pigments to the photosystems was impaired by ultraviolet radiation. Photosynthetic oxygen production was affected on a much faster timescale than changes in the absorption spectra or at the protein level.     

Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycol bis (aminoethyl ether)- N, N' -tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α-tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α-tubulin than of the β subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α-tubulin is an integral vesicle membrane protein, whereas most of the β sub-unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.     

Purification of an α-L-arabinofuranosidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α-L-arabinofurano-sidase from carrot cell cultures and its involvement in arabinose-rich polymer degradation.
An α-L-arabinofuranosidase (α-L-arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The buffer-soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE-Sephadex A-50, Sephadex G-150, Con A-Sepharose 4B and CM-Sephadex C-50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G-200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The K_m and V_max values for p-nitrophenyl α-L-arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)^-1 h^-1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca²⁺ and Zn²⁺, whereas it was strongly inhibited by Cu²⁺, Ag²⁺, Hg²⁺, p-chloromercuri-benzoate and L-arabono-l,4-lactone. The enzyme acted on beet arabinan in an exo-fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara-binogalactan and pectic polymer purified from carrot cell walls.     

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.     

ON NEUROFILAMENT and NEUROTUBULE PROTEINS FROM HUMAN AUTOPSY TISSUE

Abstract— Neurofilaments and neurotubules are the principal fibers of the mature normal neuron. In this study the protein subunits of these neurofibrils were isolated from human autopsy tissue, and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by two dimensional peptide maps of the tryptic digest of these proteins labelled with ¹²⁵I. The α and the β monomers of neurotubule are related but distinct in their peptide maps, while the major neurofilament protein subunit (molecular weight, 50,000) is remarkably similar to β tubulin. The neurofilament fraction binds colchicine, but the specificity is not yet determined. Neurotubule and neurofilament are also similar in having minor proteins which coelectrophorese on the gels. These results suggest that neurofilament and neurotubule may share one or more protein subunits.     

NADP+-dependent glutamate dehydrogenase from the facultative methylotroph Hyphomicrobium X

Abstract NADP⁺-dependent glutamate dehydrogenase (GDH; EC 1.4.1.4) was purified using acetone precipitation, heat, DEAE-cellulose and dye-ligand Ramazol Red column chromatography. The M _r of the native enzyme was estimated to be 380 000 (± 10 000) by polyacrylamide gel electrophoresis. The same technique in the presence of sodium dodecyl sulphate (SDS) gave one subunit band with an M _r of 63 400 (±4000). Thus the enzyme has a hexameric structure. The enzyme has a pH optimum of 8.5 and has K _m apparent values of 1.6 mM, 0.015 mM and 10.2 mM for α-ketoglutarate, N NADPH and L -glutamate, respectively. Michaelis-Menten kinetics were not observed when the ammonium concentration was increased. A progressive increase in the ammonium concentration resulted in a progressively increasing K _m value. The enzyme was highly specific for all substrates and markedly insensitive to inhibitors.     

Purification and characterization of tubulin from the catfishHeteropneustes fossilis

Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.