New tandem-repeating peptide structures in polysialoglycoproteins from the unfertilized eggs of kokanee salmon

New polysialoglycoproteins, designated PSGP(On), were isolated from the fertilized and unfertilized eggs of the kokanee salmon, Oncorhynchus nerka adonis. The polysialylglycan chains consisting of alpha-2,8-linked O-acetylated poly(N-glycolylneuraminyl) chains have recently been characterized. We have now determined the complete amino acid sequence of the tandem-repeating units of PSGP(On) from the unfertilized eggs of kokanee salmon and found that the following two distinct forms are present in PSGP(On) in almost identical amounts: [formula: see text] and [formula: see text] where * denotes the O-glycosylation site and mean value of m, n = about 20. Upon fertilization these high-molecular-weight forms of PSGP(On) were proteolytically cleaved to the corresponding repeating units, low-molecular-weight PSGP(On), by the action of a specific protease (PSGPase) at the position two residues set C-terminally to the Pro residue and N-terminally to the Asp residue, i.e. -Pro-Ser-Xaa-Asp-: [formula: see text] and [formula: see text].     

Comparative structures of the apopolysialoglycoproteins from unfertilized and fertilized eggs of salmonid fishes

The complete amino acid sequence of the major polysialoglycoproteins (PSGPs) from two genera of salmonid fish eggs, Salvelinus and Oncorhynchus, has been determined. The occurrence of tandem repeats of a genus-specific dodeca- and tridecapeptide was found for the apoPSGP of Salvelinus leucomaenis pluvius (Slp) and Oncorhynchus masou ishikawai (Omi), respectively, their amino acid sequences being highly homologous with that of rainbow trout [Salmo gairdneri (Sg)] apoPSGP (*denotes the glycosylation site; mean value of N = approximately 25): H-PSGP(Slp): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-)N H-PSGP(Omi): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser-)N H-PSGP(Sg): (Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly-)N Within 5-7 min following fertilization H-PSGP is converted to the low-molecular-mass PSGP (L-PSGP) by a specific protease (PSGPase). We have purified L-PSGP from the fertilized eggs of S. leucomaenis pluvius and Oncorhynchus keta (chum salmon) and compared it with rainbow trout egg L-PSGP(Sg) by analysis of their amino acid sequence: L-PSGP(Slp): Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Asp L-PSGP(Ok): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Ser L-PSGP(Sg): Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly The data support the conclusion that H-PSGP is degraded in vivo 5-7 min after fertilization to L-PSGP by proteolytic cleavage at the position two residues C-terminally to the Pro residue, i.e., -Pro-Ser-Xaa-Asp-(Xaa = either Gly, Ser, or Asp) by the action of PSGPase.     

Use of a bacteriophage-derived endo-N-acetylneuraminidase and an equine antipolysialyl antibody to characterize the polysialyl residues in salmonid fish egg polysialoglycoproteins. Substrate and immunospecificity studies

Polysialoglycoproteins (PSGP), a class of glycoproteins containing oligo(poly)sialylglycan chains, are the major glycoprotein components in cortical alveoli of a number of Salmonidae fish eggs. Lake trout, Salvelinus namaycush, egg PSGP (PSGP(Sn)) differs from rainbow trout, Salmo gairdneri, egg PSGP (PSGP(Sg)) in its sialic acid composition; the former contains both N-acetyl- and N-glycolyl-D-neuraminic acid residues, designated Neu5Ac and Neu5Gc, while the latter contains only Neu5Gc residues. Fragmentation analysis of oligo(poly)sialyl chains in lake trout PSGP(Sn) has established that there are two distinct types of oligo(poly)sialyl structures in this PSGP molecule, namely alpha-2,8-linked oligo/poly(Neu5Ac) and alpha-2,8-linked oligo/poly(Neu5Gc). No hybrid structure having both Neu5Ac and Neu5Gc residues in the fragment oligosialic acids was detected. These two distinct PSGP preparations from eggs of lake trout and rainbow trout have been used to compare their immunoreactivity with anti-polysialyl antibodies (H.46) and sensitivity to a bacteriophage-derived (Escherichia coli K1F) endo-N-acetylneuraminidase (Endo-N). H.46 was found to cross-react only with lake trout PSGP(Sn) in immunodiffusion assays but not with rainbow trout PSGP(Sg), indicating that H.46 is a specific probe for alpha-2,8-linked poly(Neu5Ac) but not for poly(Neu5Gc). In contrast, Endo-N was found to catalyze the hydrolysis of both alpha-2,8-linked poly (Neu5Ac) and poly(Neu5Gc), so that this enzyme can be used as a diagnostic reagent for detecting both types of polysialic acids. H.46 was used in indirect immunofluorescence experiments to localize PSGP(Sn) in cortical alveoli isolated from lake trout eggs.     

Localization of polysialoglycoprotein as a major glycoprotein component in cortical alveoli of the unfertilized eggs of Salmo gairdneri

Polysialoglycoprotein (PSGP, 200 kDa), first isolated by S. Inoue and M. Iwasaki in 1978 (Biochem. Biophys. Res. Commun. 83, 1018-1023) from unfertilized eggs of rainbow trout, has been shown to comprise a unique class of glycoproteins associated with the exocytosis of cortical alveoli. In 1986, 200-kDa PSGP was shown to undergo proteolytic depolymerization to 9-kDa PSGP on egg fertilization (activation) and there was an indication that 200-kDa PSGP may possibly be a component of cortical alveoli (J. Biol. Chem. 261, 5256-5261). In this paper we present evidence demonstrating that PSGP is actually a component of cortical alveolus. First, a cortical alveolus-rich fraction (CA fraction) was obtained by low-speed centrifugation of the homogenate of unfertilized eggs of rainbow trout. The 200-kDa PSGP was found to be a major component extractable with buffered saline from the CA fraction by chemical analysis of isolated materials. Treatment of the eggs to induce parthenogenetic activation resulted in all cases in the loss of both cortical alveoli and PSGP in the CA fraction. Second, perivitelline space fluid was isolated from the activated eggs of rainbow trout and analyzed, and 9-kDa PSGP was confirmed to be present as a major proteinaceous component. Third, following incubation of the eggs in water for activation, the time course of the appearance of 9-kDa PSGP and the breakdown of 200-kDa PSGP was observed. The formation of 9-kDa PSGP was detected in the eggs after 1 min of incubation and its level rose rapidly, attaining a maximum at 7 min after incubation. During this period, there was a concomitant fall in the level of 200-kDa PSGP. This formation and rapid increase in 9-kDa PSGP correspond directly to the time course of cortical alveolus exocytosis in activated chum salmon eggs recently studied by scanning electron microscopy.     

Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary

Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing alpha2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two alpha2,8-polysialyltransferases (alpha2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which alpha2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the alpha2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo alpha2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the alpha2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP.     

Polysialoglycoproteins of Salmonidae fish eggs. Complete structure of 200-kDa polysialoglycoprotein from the unfertilized eggs of rainbow trout (Salmo gairdneri)

Polysialoglycoproteins (PSGP) we first isolated from the unfertilized eggs of rainbow trout (Salmo gairderi) and now found to be a ubiquitous component of Salmonidae fish eggs are a novel type of glycoprotein. PSGP from rainbow trout has a molecular weight of 200 X 10(3), a low protein content (about 15% w/w), and a high sialic acid (N-glycolylneuraminic acid (NeuGc] content (about 60%, w/w). In any evaluation of the biological functions of PSGP, information about the complete structure of this unique macromolecular component is relevant. We have now completed the determination of the overall structural organization of the 200-kDa PSGP, and this is the first report of the complete structural analysis of this novel class of glycoprotein: (Asp)0-2-Ala-Thr*-Ser*-Glu-(Ala-Ala-Thr*-Gly-Pro-Ser-Gly-Asp-Asp-Ala-Thr *-Ser*- Glu)n-Ala-Ala-Thr*-Gly-Pro-Ser-Gly where * indicates the amino acid residues to which oligo- and/or polysialylglycan units are attached and n = 25. Thus the most outstanding structural features of PSGP isolated from the unfertilized eggs of rainbow trout are now the occurrence of (a) tandem repeats of a tridecapeptide and (b) an alpha-2----8-linked oligo(poly)sialyl group on each of the core oligosaccharide chains, i.e. GalNAc- beta 1----4(NeuGc alpha 2----3)GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n ----6)GalNAc alpha 1----Ser (or Thr), GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), and Gal beta 1----3[----8NeuGc alpha 2)n----6) GalNAc alpha 1----Ser (or Thr).     

The effect of temperature on recombination activity in testes of rodents

An extractable enzyme system capable of catalyzing recombination in vitro was described in murine spermatocytes [Hotta et al. (1985) Chromosoma 93, 140-151]. The system is specific to meiosis, its activity increasing 400-fold between the premeiotic S-phase and mid-pachytene. The present study examines the effect of temperature on this system since the elevation of testicular temperature is one of the major factors causing impairment of testicular function. A strong depression of in vitro recombination activity occurred immediately after raising the testicular temperature in vivo by translocating the testes into the abdominal cavity (cryptorchid). The in vitro study also showed that the extract from spermatocytes preferred lower temperatures (30-32 degrees C) than somatic cells (37 degrees C) for maximal activity of recombination. These results suggest that the strong depression of recombination activity may be an important factor which causes degeneration of testes by heat.     

Structures of the carbohydrate units of polysialoglycoproteins isolated from the eggs of four species of salmonid fishes

Fish egg polysialoglycoprotein (PSGP) is a novel type of 200 kDa-glycoprotein containing more than 50% sialic acid by weight and about 90O-glycosidically-linked sialoglycan units per molecule. Of about 100 different molecular species assumed to be present in a sialoglycan mixture obtained by alkaline borohydride treatment ofSalvelinus leucomaenis pluvius PSGP, 23 mono- to tetrasialylglycans were isolated by anion-exchange chromatography and preparative column chromatography on porous silica, and their structures were determined. Core asialo-oligosaccharides were obtained from PSGP of four species of salmonid fishes by exhaustive enzymatic desialylation of sialoglycan mixtures and the structures of purified compounds were determined. Two complete types of asialopentasaccharide core structures, Fuc1-3GalNAc1-3Gal1-4 Gal1-3GalNAcOL, and GalNAc1-4GalNAc1-3Gal1-4Gal1-3GalNAcOL, and all of the possible biosynthetic precursors of these pentasaccharide cores were found in every PSGP examined. All types of oligosaccharide chains, both complete and incomplete, were found to occur in highly sialylated forms in PSGP.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - GalNAcOL N-acetylgalactosaminitol - PSGP polysialoglycoprotein - SRO sialidase-resistant oligosaccharide     

Purification, characterization, and rapid inactivation of thermolabile ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85

Conjugation of ubiquitin to certain proteins can trigger their degradation in the in vitro reticulocyte system. In order to determine whether ubiquitin conjugation serves as an intermediate step in the turnover of cellular proteins in vivo, it is necessary to isolate proteolytic intermediates, i.e. ubiquitin-protein adducts of specific cellular proteins. While the steady-state level of conjugates of rapidly turning over proteins is relatively high, that of long-lived proteins is presumably extremely low, and therefore undetectable. Therefore, mutant cell lines with conditionally altered function(s) of the ubiquitin system can serve as powerful tools in studying the degradation of stable cellular proteins. We have characterized a temperature sensitive cell cycle arrest mutant cell (ts85) with a thermolabile ubiquitin-activating enzyme (E1; Finley, D., Ciechanover, A., and Varshavsky, A. (1984) Cell 37, 43-55). Following incubation at the restrictive temperature (39.5 degrees C), these cells fail to degrade short-lived proteins (Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66). However, involvement of the ubiquitin system in the turnover of long-lived proteins has not been addressed in these cells. A slow rate of inactivation of E1 in vivo, and significant rate of cell death following long incubation periods at the restrictive temperature, make this question difficult to address experimentally. In the present study we show that incubation of the cells for 1 h at 43 degrees C leads to rapid inactivation of ubiquitin conjugation in the intact mutant cell. Following heat treatment, the cells can be incubated at 39.5 degrees C for at least 6 h in order to study the possible involvement of the system in the turnover of long-lived cellular proteins. The viability of the cells is excellent at the end of the incubation. Following extraction, we have shown that inactivation occurs much more rapidly in the cell lysate in vitro than in the intact cell (t1/2 of 10 min compared to 4 h at 39.5 degrees C). The enzyme from both the mutant cell and the wild-type cell was purified to homogeneity. The molecular mass of the native enzyme from both cells is approximately 220 kDa with a subunit molecular mass of about 108 kDa. The structure of the enzyme is therefore very similar to that purified from rabbit reticulocytes. At the permissive temperature, the enzymes from both cells catalyze ATP-PPi and ATP-AMP exchange in similar kinetics. However, at the high temperature, the mutated enzyme is at least 7-fold less stable than the wild-type enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Roles of the orlA, tsE, and bimG genes of Aspergillus nidulans in chitin synthesis.

Strains of Aspergillus nidulans carrying the orlA1 or tse6 allele are deficient in cell wall chitin and undergo lysis at restrictive temperatures. The strains are remediable by osmotic stabilizers or by the presence of N-acetylglucosamine (GlcNAc) in the medium. The remediation by GlcNAc suggests that the lesion(s) in chitin synthesis resides in the amino sugar biosynthetic pathway prior to the synthesis of N-acetylglucosamine-6-phosphate. orlA1 strains grown at permissive temperature exhibit an abnormally low specific activity for L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), the first enzyme unique to amino sugar synthesis. In addition, the enzyme produced is temperature sensitive in vitro. tsE6 strains grown at permissive temperature show virtually no amidotransferase activity. This finding is consistent with an extremely labile enzyme which is destroyed by cell breakage and extract preparation. The enzyme must be active in vivo at permissive temperatures since GlcNAc is not required for growth. Thus, two structural genes (orlA and tsE) are necessary for the amidotransferase activity. bimG11 strains are temperature sensitive for a type 1 protein phosphatase involved in cell cycle regulation and arrest in mitosis. Like orlA1 and tsE6 strains, conidia from bimG11 strains swell excessively when germinated and lyse; the germlings produced are deficient in chitin content. The amidotransferase from wild-type and mutant strains is sensitive to feedback inhibition by uridine diphosphate-N-acetylglucosamine. The sensitivity of the amidotransferase from bimG11 strains is dependent on growth temperature, while that from wild-type strains is independent of temperature. The enzyme can be desensitized in vitro under conditions consistent with a protein phosphatase reaction. It is proposed that amino sugar (and chitin biosynthesis) is partially regulated by phosphorylation-dephosphorylation of the amidotransferase or a protein regulator of the enzyme.     

Separation of Oligo/Polymers of 5-N-Acetylneuraminic Acid, 5-N-Glycolylneuraminic Acid, and 2-Keto-3-deoxy-d-glycero-d-galacto-nononic Acid by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detector

A sensitive and efficient method to analyze oligo/poly-sialic acids containing α2–8-linked 5-N-acetylneuraminic acid (Neu5Ac), 5-N-glycolylneuraminic acid (Neu5Gc), and deaminated neuraminic acid (KDN) using high-performance anion-exchange chromatography (HPAEC) with a pulsed amperometric detector (PAD-2) has been developed. Using a CarboPac PA-100 column and sodium nitrate as the pushing agent, polymers in colominic acid with degree of polymerization (DP) up to 80 were separated in 68 min. A similar DP-based resolution was also obtained on a CarboPac PA-1 column. The elution ladders of the Neu5Ac, Neu5Gc, and KDN series were sufficiently different to be used as diagnostic indices. This technique was applied to identification of the sialic acid components in a polysialoglycoprotein (PSGP) sample as well as monitoring the oligo/poly-KDN-containing fractions during the purification of KDN-containing glycoprotein (KDN-gp). The maximum DPs of oligo-Neu5Gc and oligo-KDN that can be detected in PSGP and KDN-gp hydrolysates were 11 and 8, respectively. The high sensitivity of this method was demonstrated by the quantification of Neu5Ac oligomers. Distributions of the monomer and oligo/polymers in the acid and enzymatic hydrolysates of colominic acid and PSGP under different conditions were also studied.     

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Detection, isolation, and characterization of oligo/poly(sialic acid) and oligo/poly(deaminoneuraminic acid) units in glycoconjugates.

We have evaluated methods for separation, preparation, and characterization of alpha-2----8-linked oligomers of sialic acids (Neu5Ac and Neu5Gc) and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) recently found as a naturally occurring novel type of sialic acid analogue. (A) We examined preparative anion-exchange chromatography for fractionation and preparation of oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN). (B) We also examined the TLC method for separation and differentiation of the partial acid hydrolysates of colominic acid, as well as polysialoglycoproteins (PSGP) and poly(KDN)-glycoproteins (KDN-gp) isolated from rainbow trout eggs, and for discrimination of lower oligomers of Neu5Ac, Neu5Gc, and KDN. (C) We developed the high-performance adsorption-partition chromatographic method for (a) separation of monomers and oligomers of three nonulosonates according to the difference in substituents at C-5 and the presence or absence of 9-O-acetyl groups in oligo(KDN) and (b) separation of three homologous series of lower oligomers according to the degree of polymerization. (D) We examined and compared high-performance anion-exchange chromatographic separation of 3H-labeled oligo(Neu5Ac), oligo(Neu5Gc), and oligo(KDN) alditols by using Mono-Q HR 5/5 resin. (E) We examined a method of selective and quantitative microprecipitation for separation and purification of oligomers and polymers of Neu5Ac by treating them with cetylpyridinium chloride. We also used PSGP and KDN-gp to test both the sensitivity and the selectivity of this method.     

Characteristics of Methylobacillus flagellatum KT mutants, deficient for phosphoglucoisomerase, an enzyme of the ribulose monophosphate cycle of obligate methylotrophic bacteria

The activities of the key enzymes of ribulose monophosphate cycle for formaldehyde oxidation and assimilation were tested in crude extracts from temperature sensitive mutants of obligatemethylotroph M. flagellatum KT. Two mutants deficient in phosphoglucoisomerase activity were identified during this screening. Phosphoglucoisomerase of T525 pgi-1 mutant was active both at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. Complete inactivation of the enzyme at 42 degrees C occurred in 2 h in vitro, while in vivo incubation at nonpermissive temperature for more than 10 h was required for the enzyme inactivation. Phosphoglucoisomerase activity of T566 pgi-2 was 5-fold lower as compared with the one from the parent strain incubated at 30 degrees C. The enzyme was inactivated in 2 min. in crude extract at nonpermissive temperature.     

In vivo enzyme control through a strong stationary magnetic field--the case of thymidine kinase in mouse bone marrow cells

The influence of a strong homogeneous and stationary magnetic field (SMF) on the activity of the enzyme thymidine kinase (TdR-K) in bone marrow cells, and as a consequence of this on the incorporation of 125I-labelled 5-iodo-2-deoxyuridine (125IUdR) into DNA of mice and into isolated bone marrow cells in vitro, was assayed after exposure of immobilized mice. No effect could be elicited in moving mice, in cells in suspension or in enzyme in solution. The response depended on the body temperature during exposure: at 27 degrees C and 29 degrees C there was an increase and at 37 degrees C and a depression of enzyme activity. The TdR-K activity at low temperature increased with the field strength ranging from 0.2 to 1.4T. Thirty minutes were required for full expression of the effect at 1.4T; 5-10 min were needed after exposure for a return to base-line levels. Mice were given total-body irradiation at a dose of 0.1 Gy 137Cs gamma rays and then exposed immediately to a magnetic field at 1.4T for 30 min at a body temperature of 27 degrees C; gamma irradiation no longer inhibited the enzyme. Exposure to the magnetic field further removed from the time of gamma irradiation, did not negate the inhibitory effect of gamma irradiation. The observed responses to given challenges in this complex system support the hypothesis that the magnetic field affects TdR-K activity by way of a mediating structure, such as a membrane.     

W-reactivation of phage lambda in recF, recL, uvrA, and uvrB mutants of E. coli K-12.

Summary Assay conditions are described which permit detection of cryptic temperature sensitive RNA polymerases in vitro. RNA polymerase was prepared from fifteen different temperature sensitive mutants of Salmonella typhimurium chosen at random from a larger group isolated by localized mutagenesis and uridine suicide techniques. The dependence of enzyme activity on temperature, ionic strength and pH was studied in vitro. Assays at higher ionic strength (0.23 M) and temperature (50°C) distinguish three classes of mutants (Table 2). Activity of seven mutant RNA polymerases (called Class 1) under these conditions was 1% to 5% that of the parental RNA polymerase. Five mutant RNA polymerases (called Class 2) had 18% to 64% of the parental activity and three were not distinguishable from the parental enzyme under these conditions. Mixing experiments showed that the defect in Class 1 mutant enzymes is a property of the enzymes and not due to a diffusible inhibitor. In one case the lesion was shown to reside in the core enzyme. Class 1 mutant RNA polymerases were shown to be irreversibly inactivated during the assay at higher temperature and ionic strength. This suggests that the Class 1 enzymes may be more thermolabile than the wild type enzyme or may fail to be protected from thermal denaturation by formation of a ternary complex with template and product. We conclude that the method used to isolate these mutants (Young et al., 1976) and the assay described here (Table 2) are efficient ways to isolate and detect temperature sensitive RNA polymerase mutants of Salmonella typhimurium.     

Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase

We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II L-asparaginase. K(m) for L-asparagine is 17 ± 0.9 μM and pI 5.4 ± 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 °C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. colil-asparaginase (EcA) as the reference substance at similar conditions.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.