Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Precursor supply for insect juvenile hormone III biosynthesis in a cockroach

The biosynthesis of the sesquiterpenoid juvenile hormone III (JH III) was studied using corpora allata of the cockroach Diploptera punctata incubated in vitro and a radiochemical assay for the hormone produced. The influence of several exogenous precursors such as glucose, trehalose, acetate, amino acids, and mevalonate on JH synthetic rates was studied. Glucose or trehalose were needed for an optimal rate of JH synthesis. Highest rates were achieved at trehalose concentrations below the normal hemolymph levels (35-40 mM). About one-third of the glucose utilized for the biosynthesis of JH III was metabolized through a pentose pathway, but acetyl-CoA derived from glucose was significantly diluted by acetyl-CoA from other sources. Amino acids provided both a source of carbon for JH III synthesis and a source of energy that allowed JH III synthesis from acetate and stimulated JH III synthesis from glucose. Acetate was a poor substrate, because it could not support JH III synthesis in long term incubations. The incorporation of exogenous mevalonate into JH III was dependent on the physiological state of the glands, but there was a significant dilution with endogenous mevalonate. This dilution reflected in part the poor penetration of mevalonate into the corpora allata cells, because JH synthesis in mevinolin-treated cells was not fully rescued by mevalonate.     

Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia

An enzymatic system has been isolated that catalyzes dihydroxylation of phthalate to form 1,2-dihydroxy-4,5-dicarboxy-3,5-cyclohexadiene with consumption of NADH and O2. This system is comprised of two proteins: a flavo-iron-sulfur protein with NADH-dependent oxidoreductase activity and a nonheme iron protein with oxygenase activity. Phthalate oxygenase is a large (approximately 217 kDa) protein composed of apparently identical 48-kDa monomers. The active enzyme has one Rieske-type [2Fe-2S] center and one mononuclear iron/monomer. Removal of the mononuclear iron by incubation with EDTA or with o-phenanthroline inhibits oxygenation; ferrous ion completely restores activity. No other metals are effective. Phthalate oxygenase is specific for phthalate or other closely related compounds. However, only phthalate is tightly coupled to NADH oxidation and O2 consumption with a stoichiometry of 1:1:1. Phthalate oxygenase is chemically competent to oxygenate phthalate when artificially supplied with reducing equivalents and O2. Phthalate oxygenase reductase is required, however, for efficient catalytic activity. The reductase is a monomeric 34-kDa flavo-iron-sulfur protein containing FMN and a plant-ferredoxin-type [2Fe-2S] center in a 1:1 ratio. Phthalate oxygenase reductase is specific for NADH but can pass electrons to a variety of acceptors, including: phthalate oxygenase, cytochrome c, ferricyanide, and dichlorophenolindophenol. This system is similar to other bacterial oxygenase systems involved in aromatic degradation including: benzoate dioxygenase, toluene dioxygenase, benzene dioxygenase, and 4-methoxybenzoate demethoxylase. However, phthalate oxygenase can be isolated in large quantities and is more stable than most other such systems.     

Metabolic regulation during early frog development. Identification of proteins labeled by 32P-glycolytic intermediates

When 32P-labeled phosphoenolpyruvate is injected into Xenopus laevis oocytes, a 50-60-kDa protein of subunit size Mr 29,000 is rapidly labeled, followed by a second (monomeric) protein of 66 kDa concomitant with the loss of label from the first protein. We have identified these proteins as, respectively, the glycolytic enzymes phosphoglyceromutase and phosphoglucomutase. The phosphoglyceromutase is labeled at a histidine and the phosphoglucomutase at a serine, presumably at their active sites during the gluconeogenic transformation of phosphoenolpyruvate into glycogen. The transfer of the 32P label from phosphoenolpyruvate to these two enzymes also occurs in in vitro lysates made from full-grown Xenopus oocytes, eggs, or early embryos, but with a slower time course. Lysates prepared from leg muscle show labeling of the phosphoglyceromutase, but not the phosphoglucomutase, when incubated with [32P]phosphoenolpyruvate. This last result is expected in tissues showing metabolic flux largely in the glycolytic direction. The data indicate that in full-grown oocytes and embryos metabolic flux occurs largely in the gluconeogenic direction.     

The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.     

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.     

The problem of sexual inversion in the minnow, Phoxinus phoxinus (L.)

The incidence of sexual inversion in Phoxinus phoxinus has been studied by examining histologically (a) the gonads of 406 adult specimens caught from the wild during a period of 16 months, (b) the gonads of 168 adults kept in captivity in groups with an experimentally altered sex-ratio, and (c) the differentiation of the gonads in the fry.
All the adults had either testes or ovaries and no case of intersexuality was observed. For both testis and ovary, we recognized two phases, depending on the seasonal cycle: an active spring-summer phase, corresponding with the period of reproduction, and a quiescent autumn-winter phase. The ovaries showed an asynchrony in oocyte development, which is typical of species that spawn many times during the breeding season.
In P. phoxinus the ovaries are easily recognizable just one month after hatching and testes are well differentiated in 2-month-old fingerlings. Differentiation is, therefore, precocious and follows a pattern typical of gonochoristic species in which hermaphroditism never occurs.     

Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.