UDP-apiose/UDP-xylose synthase. Subunit composition and binding studies.

The UDP-apiose/UDP-xylose synthase from cell suspension cultures of parsley has been purified 1400-fold by an improved method. The ratio of apiose to xylose formed from UDP-D-glucuronic acid (UDP-GlcUA) remained constant throughout the purification procedure. Dodecylsulfate-gel electrophoresis and sedimentation equilibrium measurements showed that this enzyme preparation is composed of two proteins with molecular weights of 65000 and 86000. The two proteins which are present in a molar ratio of about 1:0.7 to 1:0.9 could not be separated by ammonium sulfate fractionation, chromatography on DEAE-cellulose at different pH-values, and on omega-aminoalkyl-Sepharose, and by gel filtration on Acrylex P-100. Each protein is composed of two apparently identical subunits. The presence of only two different subunits was confirmed by end group analysis in which glycine was found as N-terminal amino acid for the larger and lysine for the smaller protein. Crosslinking with dimethylsuberimidate gave dimers of the identical subunits but no hybrids. Separation of the two proteins was achieved on DEAE-cellulose in the presence of urea. After dialysis only the 86000-Mr protein showed enzyme activity with no significant change in the apiose/xylose ratio. However, in the absence of the 65000-Mr protein enzyme stability was decreased drastically. By equilibrium dialysis it was found that 0.5 mol UDP-GlcUA are bound per mole of 86000-Mr protein. NAD+ alone was not bound, but in the presence of UDP it was also bound in a ratio of 0.5 mol/mol catalytic protein. Experiments in which sodium borohydride was added to the enzyme incubation gave no indication that the 4-keto intermediate is bound as a Schiff base to the enzyme. Also no evidence for epimerization at C-3 of the 4-ulose intermediate prior to ring contraction to apiose was found.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

An endothelial growth-stimulating factor from salivary glands

Bovine parotid gland and mouse submaxillary gland yield substances of apparent molecular weight 86000 and 80000, respectively which, when injected into either newborn or adult mice induce a hypertrophic growth response from endothelium in numerous organs. The hypertrophic endothelium thus induced closely resembles the endothelium formed in the blood islands of early embryos.     

High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.     

Isolation and partial characterization of a D-amino acid oxidase active against cephalosporin C from the yeastTrigonopsis variabilis

Summary D-Amino acid oxidase has been isolated and purified in a simple procedure to homogeneity fromTrigonopsis variabilis. The enzyme was able to oxidatively deaminate cephalosporin C to keto adipic 7-aminocephalosporanic acid. The molecular weight of the native enzyme was estimated to be 86000. The enzyme was shown to contain two identical subunits with each subunit carrying probably one molecule of iron. The K_m values determined for the substrates cephalosporin C, D-phenylalanine, D-alanine, D-methionine and D-leucine were 13, 10, 76, 0.76 and 0.12 mM, respectively.Enzyme yield was shown to increase on growth of the organism in medium supplemented with DL-methionine.     

Adenosine 3', 5'-cyclic monophosphate-binding proteins from Trypanosoma gambiense.

Two cyclic AMP-binding proteins, not identical with regulatory subunits of protein kinases, have been isolated from Trypanosoma gambiense. The cyclic AMP receptors were separated by gel chromatography on the basis of their molecular weights. The binding constants of the high and the low molecular weight receptors for cyclic AMP were determined to be 0.4 muM and 0.6 muM, respectively. Cyclic IMP and cyclic GMP compete with cyclic AMP for the binding sites of both receptors. The cyclic AMP binding of the low molecular weight receptor was competitively inhibitied by adenine derivatives. The binding capacity of the high molecular weight receptor was enhanced about two-fold by proteolytic modification with trypsin.     

Studies on inosine monophosphate dehydrogenase. Isotope exchange at equilibrium.

Investigations on the mechanism of the IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14) reactions have been made at pH 7.0 by measuring rates of isotope exchange at chemical equilibrium with K+ maintained at a constant concentration. The results are generally in accord with the conclusions reached on the basis of the steady-state kinetic data obtained previously and confirm that there is random addition of IMP and NAD to the enzyme. The data also indicate clearly that at pH 7.0 catalysis is faster than the rate of IMP and/or XMP release which is rate limiting for the reaction sequence. The binding of IMP to the enzyme at pH 8.1 has been demonstrated to occur in the absence of both K+ and NAD and id independent of the K+ concentration.     

Inosine exists in mRNA at tissue-specific levels and is most abundant in brain mRNA.

The general view that mRNA does not contain inosine has been challenged by the discovery of adenosine deaminases that act on RNA (ADARs). Although inosine monophosphate (IMP) cannot be detected in crude preparations of nucleotides derived from poly(A)+ RNA, here we show it is readily detectable and quantifiable once it is purified away from the Watson-Crick nucleotides. We report that IMP is present in mRNA at tissue-specific levels that correlate with the levels of ADAR mRNA expression. The amount of IMP present in poly(A)+ RNA isolated from various mammalian tissues suggests adenosine deamination may play an important role in regulating gene expression, particularly in brain, where we estimate one IMP is present for every 17 000 ribonucleotides.     

Mechanism of microtubule-facilitated "fast track" nuclear import

Although the microtubule (MT) cytoskeleton has been shown to facilitate nuclear import of specific cancer-regulatory proteins including p53, retinoblastoma protein, and parathyroid hormone-related protein (PTHrP), the MT association sequences (MTASs) responsible and the nature of the interplay between MT-dependent and conventional importin (IMP)-dependent nuclear translocation are unknown. Here we used site-directed mutagenesis, live cell imaging, and direct IMP and MT binding assays to map the MTAS of PTHrP for the first time, finding that it is within a short modular region (residues 82-108) that overlaps with the IMPβ1-recognized nuclear localization signal (residues 66-108) of PTHrP. Importantly, fluorescence recovery after photobleaching experiments indicated that disruption of the MT network or mutation of the MTAS of PTHrP decreases the rate of nuclear import by 2-fold. Moreover, MTAS functions depend on mutual exclusivity of binding of PTHrP to MTs and IMPβ1 such that, following MT-dependent trafficking toward the nucleus, perinuclear PTHrP can be displaced from MTs by IMPβ1 prior to import into the nucleus. This is the first molecular definition of an MTAS that facilitates protein nuclear import as well as the first delineation of the mechanism whereby cargo is transferred directly from the cytoskeleton to the cellular nuclear import apparatus. The results have broad significance with respect to fundamental processes regulating cell physiology/transformation.     

Purification of IMP dehydrogenase from rat hepatoma 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Carbamylphosphate synthetase from Salmonella typhimurium. Regulations, subunit composition, and function of the subunits.

Carbamylphosphate synthetase was purified to homogeneity from a derepressed strain of Salmonella typhimurium by a procedure based on affinity chromatography employing immobilized glutamine. The enzyme catalyzes the synthesis of carbamylphosphate from either ammonia or glutamine together with ATP and bicarbonate. The ATP saturation curve of either nitrogen donor is sigmoidal (n equals 1.5) but the affinity for ATP is higher with ammonia. In addition to the feedback inhibition by UMP and activation by ornithine which we previously reported (1), the activity was found to be stimulated by IMP and phosphoribosyl-1-pyrophosphate. Evidence from pool measurements in enteric bacteria by others suggests that of the latter two compounds only phosphoribosyl-1-pyrophosphate is physiologically significant. All effectors regulate enzyme activity by altering its affinity for ATP. Glutamine also modulates the affinity for ATP; it is increased as glutamine concentratiions decrease, an effect that could serve to insulate the cell against major changes in carbamylphosphate synthesis in response to fluctuations in concentration of glutamine. The molecular weight of the holoenzyme was estimated to be 150,000 by sucrose density gradient centrifugation in triethanolamine and Tris-acetate buffers in which the enzyme is a monomer. In the presence of ornithine in potassium phosphate buffer, the enzyme is an oligomer with a molecular weight of 580,000. This transition has been exploited as an alternate route of purifying the enzyme to homogeneity using successive sucrose density centrifugation. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate shows that the enzyme consists of two unequal subunits with molecular weights of 110,000 and 45,000. The two subunits were separated by gel filtration in the presence of 1 M potassium thiocyanate, ATP, MgCl2, glutamine, NH4Cl, ornithine, and UMP. The heavy subunit catalyzes the synthesis of carbamylphosphate from ammonia but not glutamine. The ATP saturation curve for the separated heavy subunit is still sigmoidal (n equals 1.4 and So.5 equals 0.3 mM). The ammonia dependent activity of the heavy subunit is stimulated by the activators ornithine, IMP, and phosphoribosyl-1-pyrophosphate but is only marginally inhibited by high concentrations of UMP. The addition of the light subunit restored full ability to utilize glutamine as well as normal sensitivity to UMP. Purified subunits were used for in vitro complementation studies with strains carrying mutations in pyrA, the structural gene encoding carbamylphosphate synthetase. The results indicate that the pyrA region encodes both subunits and that the structural genes for the two polypeptides are linked. A deletion mutant lacking both subunits of carbamylphosphate synthetase also lacked any ability to synthetize carbamylphosphate from ammonia. Hence, unlike certain other bacteria, S. typhimurium does not possess a carbamate kinase.     

A freeze fracture study of the developing tegumental outer membrane of Schistosoma mansoni

The freeze fracture technique has been used to quantify changes in the integral components of the double outer membrane of Schistosoma mansoni during the 6-week period of development within the mouse. The intramembraneous particle (IMP) density on the P1 face begins to rise within 6 h of host penetration, reaches a maximum at day 4 and then falls rapidly after day 9, so that it is at a low level between 3 and 6 weeks. The E1 face IMP density follows the same course as that of the P1 face except that maximum particle density is recorded on day 1 and the counts begin to fall on day 5. The IMP density on the P2 face remains at a consistently low level throughout development. The E2 face IMP density rises gradually to a peak at day 4, when the parasites have migrated to the lungs, and remains thereafter at a similar level, so that by 6 weeks the E2 face has a higher IMP density than the other three fracture faces. The E2 face IMP show a marked increase in size on day 4. Morphological studies indicate that a different type of inclusion body makes a transient appearance in the tegument of the lung worms, and immunocytochemical techniques show the lung worms to be nonimmunogenic. It is suggested, therefore, that the E2 face IMP may represent complexes of parasite antigens and acquired host antigens. The tegumental membranes of cultured specimens have also been examined by freeze fracturing and the IMP densities compared with those obtained from in vivo parasites; the cultured schistosomula have a lower E2 face particle density than the in vivo specimens.     

Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, (Na+ +K+)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in (3H-mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated Mr = 58000), there are several minor surface glycopeptides (Mr = 76000, 86000 and 92000-100000) which are apparent extrinsic membrane components, and two surface glycopeptides (Mr = 42000 and 130000) which are intrinsic membrane components.     

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.     

Carbamoyl-phosphate synthase in Phycomyces blakesleeanus

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.     

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42.9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.