Tubulin isotypes and their interaction with microtubule associated proteins

Summary To assay the functional significance of the multiple but closely related - and -tubulin polypeptides (termed isotypes) that are expressed in mammalian cells, we have generated a number of sera that uniquely discriminate among these isotypes. These sera have been used to demonstrate that there is no subcellular sorting of either - or -tubulin isotypes among microtubules of diverse function, either in cells growing in culture or in tissues consisting of cell types that contain specialized kinds of microtubule. In spite of this failure to segregate between functionally distinct kinds of microtubule, the fact that isotype-specific amino acid sequences have been strictly conserved over extensive periods of evolutionary time argues persuasively for a functional role for the different tubulin gene products. One possibility is that they are required for specific interactions with microtubule associated proteins (MAPs), and that tubulin isotypes have coevolved with different cell type-specific MAPs with which they must interact. We have tested this hypothesis by examining the distribution of -tubulin isotypes in mammalian cerebellum in relationship to the known patterns of expression of a number of MAPs, and find that these patterns correlate in the case of M 2 and MAP 3, and M 6 and MAP 1 a. These data, plus emerging data based on a structural analysis of tau, MAP 1 b and MAP 2 obtained via sequence determination of cloned cDNAs, are discussed in terms of the possible functional significance of tubulin isotype/MAP interactionsin vivo.     

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.     

In vitro test of self-incompatibility in Malus domestica

Summary An in vitro assay in which self-incompatible pollen of Malus domestica is selectively inhibited is described. This assay involves heat-labile substances diffusing from the stylar tissues — in particular, glycoproteins found in the protein extract of styles. In the presence of the self-style extract, a dramatic decrease in total protein concentration in the culture medium was revealed at 30-min germination. Pretreatment of the self-pollen with 100 mM glucose prevented this drop in protein level; moreover, tube growth was entirely restored. A possible explanation in terms of protein-carbohydrate complementation is suggested.     

Nuclear protein transitions in cuttle-fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.     

Human renal clear cell carcinoma: Establishment and characterization of a new cell line (G-2101)

Summary A human cell line has been established from a renal adenocarcinoma rib metastasis of a 58-y-old male. This cell line has been maintained in continuous culture for 20 mo. through more than 50 passages. It displays simulataneous expression of the intermediate filaments cytokeratin and vimentin. Flow cytometric analysis of DNA content reveals a major hyperdiploid population. This work was supported in part by a grant from Triton Biosciences, Inc.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Regulation of ACV synthetase activity in the beta-lactam biosynthetic pathway by carbon sources and their metabolites

The activity of -l--aminoadipyl)-l-cysteinyl-d-valine catalysis. Addition of l-cysteine to fermentation media increased -lactam production in both organisms and alleviated the negative carbon source regulation by glycerol in S. clavuligerus.     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of Acinetobacter strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly--hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly--hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P_i). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P_i). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P_i concentrations below the detection limit (<10 M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly--hydroxybutyric acid - PP_n Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 mol product formed · min^-1