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U Z Littauer

Publications and source records attributed to U Z Littauer.

At least 19 recordsLinked to original sources
PubMed1991

The 93-kDa glycine receptor-associated protein binds to tubulin.

A peripheral membrane protein with a relative molecular mass of 93,000 Da is associated with cytoplasmic domains of the inhibitory glycine receptor of mammalian spinal cord. Here, evidence is given that this 93-kDa protein binds to polymerized tubulin. First, tubulin cofractionated with the 93-kDa protein upon affinity purification of the glycine receptor. Second, tubulin bound to the isolated 93-kDa protein in an overlay procedure. Third, in assays containing the purified glycine receptor, the 93-kDa protein as well as the glycine receptor alpha and beta subunits coassembled with tubulin and microtubules. The interaction of the 93-kDa protein with tubulin displayed high affinity (KD approximately 2.5 nM) and significant cooperativity (Hill coefficient approximately 2.1) and approached a stoichiometry of approximately 1:4 under saturating conditions. These data suggest that the 93-kDa protein anchors the glycine receptor at postsynaptic sites via binding to subsynaptic tubulin.

Animals↗
PubMed1990

Characterization and intracellular distribution of microtubule-associated protein 2 in differentiating human neuroblastoma cells.

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.

Animals↗
PubMed1979

Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.

Animals↗
PubMed1979

Functionally impaired tRNA from ethionine treated rats as detected in injected Xenopus oocytes.

Treatment of rats with ethionine was found to cause severe impairment in the aminoacylation capacity of tRNA. This effect was only observed when assayed in injected oocytes, while invitro assays of aminoacylation failed to detect differences between normal tRNA and tRNA from ethionine treated animals. The effect of ethionine on the tRNA population was not uniform and differed for various amino acid specific tRNAs. Thus liver tRNA from ethionine treated rats showed a decreased capacity for phenylalanine aminoacylation, while no change was found in the case of leucine. On the other hand, the level of histidine aminoacylation was higher for tRNA from ethionine treated animals. An even more complex response was observed with methionine aminoacylation where tRNA from ethionine treated animals showed an initially faster rate than control tRNA. With more prolonged incubation periods, the methionyl-tRNA from ethionine treated animals was deacylated at an accelerated rate while the level of normal methionyl-tRNA remained almost constant. In addition to the aminoacylation reaction, the participation of aminoacyl-tRNA in protein synthesis was severely impaired. In this case, both the injected oocyte system and the cell-free wheat germ assay revealed these differences which were manifested with various mRNA and viral RNA preparations.

Amino Acyl-tRNA Synthetases↗
PubMed1978

A cationic hydroxysuccinimide ester. A reagent for labeling exterior membrane proteins.

3/-labeled N,N,N,trimethylamino-beta-alanyl-N-hydroxysuccinimido ester ([3H]TMAS), a new cationic membrane reagent, was synthesized. TMAS was shown to be impermeant through human erythrocyte membranes. Under mild physiological conditions TMAS reacted primarily with amino groups of the membrane proteins and lipids. The pattern of erythrocyte proteins labeled with [3H]TMAS was examined by polyacrylamide gel electrophoresis under denaturing conditions. Externally oriented labeling of intact erythrocytes revealed a major radioactive protein with an apparent molecular weight of 90,000. By labeling ghost preparations with [3H]TMAS the radioactivity incorporated into all the major Coomassie Brilliant Blue bands resolved by gel electrophoresis. The agreement between the results obtained with anionic and cationic amino reactive probes indicates that the ionic character of the reagent has a minor effect on the pattern of labeled exterior polypeptides observed in erythrocytes.

Affinity Labels↗
PubMed1978

Induction of differentiation in mouse neuroblastoma cells.

In the presence of 1--2% dimethyl sulfoxide (DMSO), mouse neuroblastoma cells are induced to differentiate morphologically as well as electrically. In addition, treatment of neurolbastoma cells with 2% DMSO results in a marked increase in the veratridine-activated K+ or Rb+ efflux. At 4% DMSO, neurite outgrowth is completely repressed and electrical activity is poorly developed. However, at this concentration, the cells have a relatively high resting potential which suggested that membrane components determining passive and active permeability properties are not necessarily under coordinated control. Induction of differentiation by 2% DMSO is also accompanied by an increase in a heavier molecular form of acetylcholinesterase sedimenting at 10.5S. The effect of other agents on the growth and differentiation of neuroblastoma cells is also presented.

Acetylcholinesterase↗
PubMed1977

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.

Chromatography, Affinity↗
PubMed1977

Synthesis of tubulin and actin by neuronal and glial nuclear preparations from devloping rat brain.

A system was established in which nuclear preparations from rat brains were capable of protein synthesis under cell-free conditions. The electrophoretic pattern of the synthesized proteins was similar to that found in vivo provided that the reaction mixture contained pH 5 precipitated factors derived from the high speed supernatant fraction of brain. In the absence of the pH 5 factors, using nuclear preparations from brains of 2-day-old rats, approximately 1.5% and 2% of the newly synthesized proteins were identified as tubulin and actin, respectively. In the presence of pH 5 factors, protein synthesis was stimulated and the proportion of the newly synthesized tubulin and actin increased to 26% and 11%, respectively. In contrast to nuclear fractions from 2-day-old rats, when nuclei from brains of 1-month-old rats were tested in the presence of pH 5 factors, the proportion of tubulin and actin synthesized was lower and amounted to 10% and 4%, respectively. The age-dependent change in the relative amount of the tubulin and actin synthesized is in good agreement with the translational pattern shown by brain polyribosomes in a brain cell-free system as well as with the pattern obtained with brain mRNA translated in a wheat germ cell-free system. Nuclei enriched for either neuronal or glial populations synthesized similar proportions of tubulin and actin in vitro. We conclude that the reduction in the synthesis of tubulin and actin during the postnatal development of the rat brain occurs in both neuronal and glial cells.

Actins↗
PubMed1977

Decrease in levels and rates of synthesis of tubulin and actin in developing rat brain.

The cytoplasmic and particulate tubulin content of postnatal rat brains was determined at various stages of development. The amount of tubulin in the soluble fraction was found to increase after birth and levels off at the age of 10-15 days, while the total protein content is still increasing. Indeed, the percentage of tubulin in the soluble fraction is about 33% at birth, stays at this value until day 10, and then decreases to 20% between days 10 and 15. On the other hand, the rate of increase in the level of the particulate tubulin parallels that of the total particulate proteins, and hence there is no change in the percentage of particulate tubulin during brain development. There was close agreement between the tubulin values obtained by the [3H]-colchicine binding assay and those obtained by electrophoretic resolution in sodium dodecylsulfate-polyacrylamide gels. Polyacrylamide gel electrophoresis was also utilized to determine actin levels in developing brains. The percentage of cytoplasmic brain actin also decreased with the age of the rats, from a value of 20% at birth to 10% at day 30, while the percentage of the particulate actin remained constant. The decline in the percentage of cytoplasmic tubulin and actin during brain development can be accounted for by reduction in the proportions of the respective mRNA species. Translation of poly (A)-rich brain mRNA in a wheat-germ cell-free system showed that the percentages of tubulin and actin synthesized decreased gradually with age. Similar results were obtained by analyzing the proteins produced by isolated brain polysomes in a brain cell-free system.

Actins↗