α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Purification and characterization of alpha-galactosidase from sunflower seeds

From 100 g sunflower seeds, 1.2 mg purified -galactosidase was obtained with an overall yield of 51%. The -galactosidase acted on both terminal -galactosyl residues and side-chain -galactosyl residues of the galactomanno-oligosaccharides and galactomannans. The cDNA coding for sunflower -galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40263. Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence. The deduced amino acid sequences of mature enzyme and -galactosidases from coffee, guar and Mortierella vinacea -galactosidase II showed over 81%, 77%, and 47% homology, respectively. These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups.     

In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with a α-galactosidase gene cloned from senna

Galactomannans are composed of a 1to4 mannan backbone with varying degrees of 1to6 galactose substitutions and are found in the cell walls of legume endosperm. Like other cell wall polysaccharides, many factors controlling the metabolism of galactomannans remain to be elucidated. In the endospermous legume senna (Senna occidentalis) increased -galactosidase activity has previously been observed to coincide temporarily with a decrease of the galactose content of the galactomannan. To evaluate the potential role of -galactosidase for the control of the final galactose content, a -galactosidase gene expressed in immature senna seeds was cloned and transformed into the related high-yielding species guar (Cyamopsis tetragonoloba). The isolated cDNA encoded a 406 amino acid protein with a calculated molecular mass of 44313 Da. The amino acid sequence was 75% identical to the galactomannan hydrolysing -galactosidases from germinating guar and coffee bean. The senna -galactosidase gene was inserted behind a wheat high-molecular-weight glutenin promoter in the vector employed for transformation of guar by Agrobacterium tumefaciens-mediated gene transfer. About 30% of the guar transformants produced endosperm with galactomannans where the galactose content was significantly reduced. After self-fertilization of primary transformants displaying the highest galactose reduction of the galactomannan, endosperms of R₁ plants were analysed demonstrating that this property was inherited stably to the progeny and that it was 100% coupled to the presence of the senna -galactosidase gene. This suggests that -galactosidases can be involved in the determination of the final galactose content of endosperm galactomannans, demonstrating that cell wall polysaccharide biosynthesis can be modified in vivo.     

Spontaneous mutations changing the raffinose metabolism ofLactobacillus plantarum

Lactobacillus plantarum ATCC 8014 grew poorly on raffinose agar plates, but large mutant colonies appeared in high frequency from a thin film of background growth. The -galactosidase and -galactosidase activities ofL. plantarum ATCC 8014 and a mutant strain were studied in static cultures and pH-controlled fermenter cultures. Both -galactosidase and -galactosidase production were inducible in the parental strain; the induction was not needed in the mutant. The -galactosidase activity of both strains was repressed by glucose but not by -methyl-D-glucoside. The mutant phenomenon might be an obstacle in connection to traditionalLactobacillus identification by means of carbohydrate fermentation.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Structure and properties of aCephalosporium acremonium α-galactosidase

The amino acid and sugar composition of the enzyme protein, the effect of urea, sodium dodecyl sulphate and Concanavalin A on the purified -galactosidase (EC 3.2.1.22) from the moldCephalosporium acremonium has been studied. The results obtained by gas liquid chromatography indicated the presence ofN-acetylglucosamine, mannose, galactose andN-acetylneuramic acid in the molar proportions 27311. The presence of two types of Asn-linked oligosaccharide structures in the enzyme molecule is assumed. The -galactosidase liberates (1–3), (1–4) and (1–6)-linkedd-galactose units from various synthetic and natural substrates which have been tested. The effects of pH, substrate concentration and temperature on the catalytic activity of the enzyme are described. The purified -galactosidase also exhibited a lectin activity with an affinity towards glucose, and to some extent mannose.Abbreviations p-NPG p-nitrophenyl--d-galactopyranoside - 4-MUG 4-methylumbelliferyl--d-galactopyranoside - HU hemagglutinin unit - PBS phosphate buffered saline - SDS sodium dodecyl sulphate - ConA Concanavalin A - WGA wheat germ agglutinin - LCA Lens culinaris agglutinin - PHA phytohemagglutinin fromPhaseolus vulgaris     

Characterization of α-galactosidase isoenzymes in normal and fabry human-Chinese hamster somatic cell hybrids

Summary The -galactosidases in normal man-Chinese hamster somatic cell hybrids were investigated with antibodies specific for human -galactosidase A and antibodies specific for Chinese hamster -galactosidase. It was found that an isoenzyme in hybrid cells, which has an electrophoretic mobility between that of human -galactosidase A and Chinese hamster -galactosidase, contains immunologic determinants of both human and Chinese hamster origin, suggesting that it is a heteropolymeric molecule. Moreover, the locus for human -galactosidase, which was found to be X-linked, is the locus coding for -galactosidase A. Hybrids isolated after fusion of Chinese hamster cells with cells of a patient with Fabry's disease did not express human -galactosidase A or the heteropolymeric molecule even in the presence of the active human X chromosome, indicating that the deficiency of -galactosidase A in Fabry's disease is probably due to a mutation in a structural gene resulting in the inability to form immunologically detectable and functionally active molecules of -galactosidase A.     

Membrane transport ofp-nitrophenyl-α-galactoside by the melibiose carrier ofEscherichia coli

Summary p-Nitrophenyl--galactoside (-pNPG) was found to be a substrate for the melibiose transport system ofEscherichia coli. This sugar enters induced cells via the carrier and is split by -galactosidase to galactose andp-nitrophenol. In mutant cells lacking the -galactosidase [³H]--pNPG accumulated to concentrations 15 times higher than the external medium. The transport of -pNPG is inhibited by both Na⁺ and Li⁺. Na⁺ (10mm) reduced the K_m for -pNPG from 0.45 to 0.18mm and reduced theV _max from 6.7 nmoles/min/mg dry wt to a value of 3.0.     

A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of -linkages between mannose or galactose and serine/threonine residues by -mannosidase and -galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that -mannosidase and -galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.     

Construction and expression of a new fusion protein,thymosin <Emphasis Type="Italic">α</Emphasis>1–cBLyS, <Emphasis Type="Italic">E. coli</Emphasis>

A fusion thymosin 1-soluble B lymphocyte stimulator (TM1–cBLyS) gene was generated to engineer a bifunctional lymphokine, which was then over-produced in Escherichia coli. The molecular weight of the expressed fusion protein was approximately 28kDa. After being purified by Ni-NTA affinity column, the fusion protein had full activity of BLyS with a slightly higher immunological action than synthetic TM1. Because TM1 regulates the cellular immune response and cBLyS amplifies the humoral response, this bifunctional lymphokine could be useful in the treatment of various immunodeficiency syndromes and serve as an immunomodulator to enhance the hosts response to vaccination. Revisions requested 15 October 2004; Revisions received 26 November 2004;     

Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae

Summary The constuction of two fused genes is described. One involves the in-frame fusion of the yeast prepro--factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH₂-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the -factor promoter, expressed active -galactosidase in haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF1-lacZ fusion junction provided significantly higher levels of -galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.A portion of this work appeared in Biotechnology Progress (Das and Shultz 1986) as proceedings of the symposium on Industrial Scale Protein Purification, held at the annual meeting of the Institute of Chemical Engineers in Miami Beach, Fla, USA on November 4, 1986     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

Requirement of the newly recognized ilvJ gene for the expression of a valine transaminase activity in E. coli K-12

Summary The isoleucine--ketoglutarate and valine--ketoglutarate transaminase activities have been attributed to an enzyme coded in E. coli K-12 by the ilvE gene. I report here evidence that these two activities can be dissociated and appear to be the products of two different genes. Mutants altered in the ilvE gene are devoid of isoleucine--ketoglutarate transaminase activity and possess a normal valine--ketoglutarate transaminase activity. I describe here mutants lacking valine transaminase activity. They are altered in a gene, ilvJ, located between ilvE and ilvD at 83 min on the E. coli K-12 map. Temperature-sensitive revertants of the mutant containing the ilvJ mutation show a temperature-sensitive valine--ketoglutarate transaminase activity. I conclude that ilvJ is the structural gene for valine--ketoglutarate transaminase.     

The sialyl-α2,6-lactosaminyl-structure: Biosynthesis and functional role

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found ,3- or ,6-linked to galactose (Gal), ,6-linked to N-acetylgalactosamine (GalNAc) or ,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in ,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by -galactoside ,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAc,6Gal1,4GIcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-,6-lactosaminyl structure.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.