Binding of kidney nuclear proteins to the 5′-flanking region of the rat gene for Ca2+-binding protein regucalcin: Involvement of Ca2+/calmodulin signaling

Ca²⁺-binding protein regucalcin is expressed in the kidney cortex of rats, as assayed by Northern blot analysis. The existence of kidney nuclear factor which binds to the 5'-flanking region of the rat regucalcin gene was investigated. When nuclear extracts obtained from the kidney cortex of rats were used in gel mobility-shift assays, two protein-DNA complexes were uniquely formed with the DNA fragment containing the 5'-flanking region of the rat regucalcin gene. Competition gel shift experiments indicated the specific binding region of kidney cortex nuclear proteins in the 5-flanking region of the rat regucalcin gene. The two nuclear protein-DNA complexes were formed with the same mobility in rat kidney cortex and liver, which possess detectable amounts of regucalcin mRNA in Northern blot analysis. The binding activities of nuclear factors from kidney cortex to the 5-flanking region of the rat regucalcin gene were inhibited by a single intraperitoneal administration of trifluoperazine, an antagonist of calmodulin, to rats. The present study demonstrates that kidney cortex nuclear proteins specifically bind to the 5-flanking region of the rat regucalcin gene, and that the binding activity may be partly mediated through the Ca²⁺/calmodulin-dependent process.     

Tissue-specific binding of nuclear factors to the 5′-flanking region of rat gene for calcium-binding protein regucalcin

The existence of nuclear factors which bind to the 5-flanking region of calcium-binding protein regucalcin gene in rats was investigated. We previously reported that rat regucalcin mRNA is expressed in a highly tissue-specific manner; the mRNA was mainly present in the liver but only slightly in the kidney. When the nuclear proteins extracted from the liver and kidney of rats were used in the gel mobility shift assays, a protein-DNA complex was uniquely formed with the DNA fragment containing the upstream region from the first exon of rat regucalcin gene. On the other hand, this complex was not found by using the nuclear extracts from rat brain, spleen, and heart. The nuclear proteins of these extracts, however, could specifically bind to the DNA fragment containing the first exon region of rat regucalcin gene, although Northern blot analysis did not show detectable amount of regucalcin mRNA levels in rat brain, spleen, and heart. The present study demonstrates that the existence of nuclear protein components which bind to the regucalcin gene. These identified components may be involved in the tissue-specific regulation of regucalcin gene expression.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, -actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic -actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

Hepatic over-expression of peroxisome proliferator activated receptor γ2 in the ob/ob mouse model of non‐insulin dependent diabetes mellitus

The binding of nuclear factor on the promoter region of the regucalcin gene and the expression of regucalcin in the kidney cortex of rats was investigated. Nuclear extracts from kidney cortex were used for oligonucleotide competition gel mobility shift assay. An oligonucleotide between position –523 and –506 in the 5-flanking region of the rat regucalcin gene, which contains a nuclear factor I (NF1) consensus motif TTGGC(N)₆CC, competed with the probe for the binding of the nuclear protein from kidney cortex. The mutation of TTGGC in the consensus sequence caused an inhibition of the binding of nuclear factors. The binding of nuclear factor on the 5-flanking region was clearly reduced in the kidney cortex obtained at 1, 2, and 3 days after a single intraperitoneal administration of cisplatin (1.0 mg/100 g body wt) to rats. Moreover, cisplatin administration caused a remarkable decrease in regucalcin mRNA levels and regucalcin concentration in the kidney cortex. Also, serum regucalcin concentration was significantly decreased by cisplatin administration. Meanwhile, serum urea nitrogen concentration was markedly elevated by cisplatin administration. The present study demonstrates that the specific nuclear factor binds to the NF1-like sequence in the promotor region of regucalcin gene in the kidney cortex of rats, and that the nuclear factor binding and regucalcin expression are suppressed by cisplatin administration.     

Expression of calcium-binding protein regucalcin mRNA in the cloned rat hepatoma cells (Hr-II-E) is stimulated through Ca2+ signaling factors: Involvement of protein kinase C

The expression of hepatic Ca²⁺-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 × 10^-6 M), a Ca²⁺ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10^-3 M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10^-6 M) or estrogen (10^-8 M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 × 10^-9 M) or dexamethasone (10^-6 M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10^-5 M), an antagonist of calmodulin, or staurosporine (10^-7 M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca²⁺-dependent signaling factors.     

In vitro translation of rat liver and Novikoff hepatoma cytokeratin mRNAs

Summary Cytoplasmic poly(A)⁺ RNA was isolated from normal rat liver and Novikoff ascites hepatoma cells, translated in vitro using rabbit reticulocyte lysate system and the translational products were assayed by immunoprecipitation with antibodies specific for Novikoff hepatoma principal cytokeratins p39, p49 (a group of hepatic cytokeratins C, D, and E) and p56. The identity of the precipitated antigens was further confirmed by two-dimensional polyacrylamide gel electrophoresis. Only the Novikoff hepatoma poly(A)⁺ RNA contained translatable mRNA coding for the p39 cytokeratin while the p49 and p56 cytokeratins were translated from both the normal rat liver and Novikoff hepatoma poly(A)⁺ RNAs. Immunoprecipitations employing monoclonal antibody specific for p39 also recovered significant quantities of p56 and 49K cytokeratins, presumably due to oligomeric associations of these proteins with p39 immediately after in vitro synthesis. Similar results were observed after experiments with anti-p56 monoclonal antibody in which p39, not reactive with this antibody, was recovered in immunoprecipitates. Overall, the two-dimensional gel fluorograms of cytokeratins synthesized in vitro from NAH or liver poly(A)⁺ RNA are quite similar to isolated antigenic and cytokeratin profiles reported previously. These results suggest that overt posttranslational processing is not likely responsible for the diversity of cytokeratins observed in the liver.Abbreviations NAH Novikoff ascites hepatoma - HEPES N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Inverse relationship between poly (ADP-ribose) polymerase activity and 2′,5′-oligoadenylates core level in estrogen-treated immature rat

Summary The activity of poly (ADP-ribose) polymerase (ADPRP) and the content of 2,5-oligodenylates core (2,5A_n; n = 2,3 and 4) were measured in homogenates of the uterus and of the liver of immature rats immediately before (time 0) or at different times after injection of estradiol-valerate. ADPRP activity increased gradualy, starting 6 hours after estrogen injection, for about 4 days. Instead, the content of 2,5 A_n decreased by about 50% within 6 hours, and thereafter more slowly for 4 days to about 20% of starting values. Estrogen increased ADPRP activity and decreased 2,5A_n concentration also in the kidney and in the cardiac muscle of the same animals, but not in the skeletal muscle, where neither of the two parameters was affected. Injection of vehicle only (sesame oil) had no effect on ADPRP activity nor on 2,5A_n content of immature rat tissues.     

17β-Estradiol stimulates the expression of hepatic calcium-binding protein regucalcin mRNA in rats

The effect of nuclear receptor-related hormones on the expression of hepatic calcium-binding protein regucalcin mRNA in rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb of open-reading frame). A single subcutaneons administration of 17-estradiol (0.5, 1.0 and 2.0 mg/kg body weight) in rats induced a remarkable increase of regucalcin mRNA in liver; the level was about 200% of control at 24 h after the administration of 2.0 mg/kg. The increase showed about 350% even at 6 h after the administration. Meanwhile, hepatic regucalcin mRNA level was not appreciably altered by a single subcutaneous administration of thyroxine (T₄) (20, 40 and 80 mg/kg) or hydrocortisone (10 and 30 mg/kg) in rats. The present study demonstrates that the expression of hepatic regucalcin mRNA is stimulated by estrogen action in the liver nuclei of rats.     

Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector

The pharmaceutically important plant, licorice (Glycyrrhiza uralenesis Fisher), was transformed with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. The transgenic state of transformed roots was confirmed by detection of agropine and mannopine and by Southern blot hybridization with T-DNA of pGSGluc1. One to three copies of T-DNA of pGSGluc1 was integrated into the genomic DNA of G. uralensis. The expression of chimeric neo and gus genes driven by TR 1 and 2 promoters, respectively, was demonstrated by enzymatic assays. Histochemical analysis showed that the chimeric TR2-gus gene was expressed specifically in phloem and pericycle tissues of the transformed licorice roots.Abbreviations NPT-II neomycin phosphotransferase II - neo NPT-II gene from Tn5 - GUS ß-glucuronidase - gus GUS gene from Escherichia coli - TR 1–2 genes 1 and 2 of TR-DNA of pTiAch5 - Rif rifampicin     

Actin genes expressed during early development ofPatella vulgata

Summary The actin gene family of the marine molluscPatella vulgata was chosen as a model system to study the regulation of genes expressed during early development in molluscs. Using a hamster actin cDNA clone as a probe, we isolated nine actin cDNA clones from trochophore larvae. The total nucleic acid sequence of three of these clones has been determined. Each clone contains the whole protein encoding region. The deduced amino acid sequences resemble actin proteins from other species to a high extent. The nucleotide sequence from the 3UTR (UnTranslated Region) and 5UTR from all nine clones has been resolved. In this way we could identify four different subtypes. Southern blots with genomic DNA were probed with different 3UTR's corresponding to each subtype to determine the genomic organization. One 3UTR detected one band probably corresponding with one gene. Another 3UTR detected one or two genes and the third 3UTR between two and four genes. Northern blots were used to detect the presence of actin mRNA during different stages of development. In the mature oocyte, actin mRNA is present in low amounts. The level of actin mRNA starts to rise steadily from 8 h after fertilization (88-cell stage) onwards. The level of the different subtype mRNAs, as specified by their 3UTR rises at different developmental stages and to various extents. This indicates that the expression of each type is regulated independently and in relation to the developmental stage of the embryo. Correspondence to: A.E. van Loon     

Functional analysis of cis-elements, auxin response and early developmental profiles of the mannopine synthase bidirectional promoter

Summary The dual MAS1-2 promoter regulating two divergently transcribed mannopine synthase genes has been widely employed in plant expression vectors. As part of an effort towards its rational design as a genetic engineering tool, we have undertaken a functional analysis of the promoter by deletion mutagenesis and by the use of hybrid promoter constructs. Our results indicate that the central region of the intergenic promoter is composed of at least four domains. Three of these contain complementary sequences, which can potentially hybridize to form alternative palindromic structures. These three domains can function cooperatively, and in an orientation-independent manner, in imparting a sevenfold higher expression level at the 2 end relative to the corresponding 1. The remaining domain is characterized by tracts of repeated A/T-rich elements, and appears to confer the weak activity at the MAS1 promoter end. However, even though this A/T-rich DNA segment is functional, our deletion analysis provided strong evidence that it is completely dispensable for wild-type promoter activity. In addition, the relative distances between these enhancer domains and the 1–2 TATA-proximal regions can have a pronounced influence on the level of expression in both directions. In young tobacco seedlings, the two promoter ends are expressed in similar, if not identical, tissues in the aerial parts of the plants, but major differences can be observed in roots. Transient expression assays using hybrid promoter constructs showed that cis-elements that can respond to auxin induction signals are redundant in nature, in that they are dispersed throughout the promoter and showed no obvious consensus sequence.     

Overexpression of the 3'-untranslated region of myelin proteolipid protein mRNA leads to reduced expression of endogenous proteolipid mRNA

The current studies focus on what mechanisms regulate the concentration of PLP mRNA in cells. The PLP mRNA is very stable and these studies suggest that its stability is regulated by a trans-acting factor specific to oligodendrocytes. In order to test whether the 3untranslated region (3UTR) of the PLP mRNA might regulate PLP RNA stability, C6 cells were transfected with cDNAs that expressed either luciferase or luciferase fused to the 3UTR of PLP. Although transgene expression was low, in cells transfected with the PLP 3UTR, there was a significant decrease in the endogenous PLP mRNA. These cells showed a distinct change in morphology and in adhesion properties. Thus, there may be a role for plp gene products in cell adhesion, which was downregulated in these cells, or an unknown function may be encoded by the PLP 3UTR. Transgenic mice that overexpress enhanced green fluorescent protein fused to the PLP 3UTR under control of PLP regulatory sequences were tested for the expression of the endogenous PLP mRNA. Three of four lines of transgenic mice had decreased endogenous PLP mRNA, relative to their non-transgenic littermates; the EGFP-PLP 3UTR mouse line that expressed the highest level of transgene mRNA had a 54% reduction in PLP mRNA. We hypothesize that the PLP mRNA is regulated by elements in the 3UTR and stabilizing proteins specific to oligodendrocytes, and that in cells that overexpress the PLP 3UTR, these stabilizing proteins may be insufficient to maintain the normal level of the endogenous PLP mRNA.     

Effects of cold exposure on cyclic AMP concentration in plasma,liver, and brown and white adipose tissues in cold-acclimated rats

Effects of acute cold exposure on plasma energy substrates and tissue 3,5-adenosine monophosphate (cAMP) were analyzed in intact rats, to define an involvement of the nucleotide in nonshivering thermogenesis (NST) and resultant cold acclimation. After an acute cold exposure to –5°C, the plasma glucose level increased gradually in warm-kept control rats (C) while it decreased significantly in cold-acclimated rats (CA). However, it was increased considerably by an extreme cold exposure to –15°C in both C and CA. By contrast, plasma levels of free fatty acids (FFA) increased immediately after cold exposure and the release lasted during the period of exposure especially in C. The cold exposure also increased plasma cAMP concentration but no concomitant increase was found in the liver. In both brown (IBAT) and white (WAT) adipose tissues the nucleotide concentration showed a stepwise decrease. The observed correlation between lipolysis and plasma cAMP response after cold exposure suggests an involvement of the adenylate cyclase-cAMP system in NST via lipid metabolism, at least, in the early stages of cold acclimation.Abbreviations cAMP cyclic 3,5-adenosine monophosphate - NST nonshivering thermogenesis - FFA free fatty acids - IBAT brown adipose tissue - WAT white adipose tissue     

Differential response of the NADH oxidase of plasma membranes of rat liver and hepatoma and HeLa cells to thiol reagents

NADH oxidase activity of plasma membranes from rat hepatoma and HeLa cells responded to thiol reagents in a manner different from that of plasma membranes of liver. Specifically, the NADH oxidase activity of plasma membranes of HeLa cells was inhibited by submicromolar concentrations of the thiol reagentsp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), or 5,5-dithiobis-(2-nitrophenylbenzoic acid) (DTNB), whereas that of the rat liver plasma membranes was unaffected or stimulated over a wide range of concentrations extending into the millimolar range. With some hepatoma preparations, the NADH oxidase activity of hepatoma plasma membranes was stimulated rather than inhibited by PCMB, whereas with all preparations of hepatoma plasma membranes, NEM and DTNB stimulated the activity. In contrast, NADH oxidase activity of rat liver plasma membrane was largely unaffected over the same range of PCMB concentrations that either stimulated or inhibited with rat hepatoma or HeLa cell plasma membranes. Dithiothreitol and glutathione stimulated NADH oxidase activity of plasma membranes of rat liver and hepatoma but inhibited that of HeLa plasma membranes. The findings demonstrate a difference between the NADH oxidase activity of normal rat liver plasma membranes of rat hepatoma and HeLa cell plasma membranes in addition to the differential response to growth factors and hormones reported previously (Brunoet al., 1992). Results are consistent with a structural modification of a NADH oxidase activity involving thiol groups present in plasma membranes of rat hepatoma and HeLa cells but absent or inaccessible with plasma membranes of rat liver.     

Evolutionary conservation of the 3′ ends of members of a family of giant secretory protein genes inChironomus pallidivittatus

Summary Two cDNA clones representing the 3-end regions of BR1 and BR2 75S mRNA were obtained fromChironomus pallidivittatus. The regular structure characterizing the core of these genes, consisting of tandemly arranged repeat units, changes into a more irregular structure toward the 3 end. Distal to a standard type of repeat unit with a characteristic excess of positive charges, a new type of repeat with a high, negative charge density is interspersed among parts of the standard unit. The last 111 amino acids before the stop codon represent a unique region distinctly different in amino acid composition from upstream regions, and include two partially homologous hydrophobic regions. Sequence comparison of 3-end regions from clones representing BR1 and BR2 genes indicates striking sequence conservation in the unique part of the region. Analysis of the level of silent site divergence shows that the homology increases in the 3 direction up to the polyadenylation site. That the unique region is retained as a part of the secreted protein is shown by Western blotting.     

Tissue concentration of calcium-binding protein regucalcin in rats by enzyme-linked immunoadsorbent assay

The concentration of calcium-binding protein regucalcin in the tissues of rats was estimated by enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG. In male rats (5 weeks old), regucalcin was most pronounced in the liver. Liver regulcalcin concentration was about 0.1M, when it was calculated with regucalcin molecular weight of 28,800. The relatively higher level of regucalcin was also found in the kidney as compared with that of the skeletal muscle, duodenum, testis, lung, heart, spleen, cerebral cortex and hippocampus. Similarly in female rats, regulacalcin was remarkable in the liver, and appeared only slightly in the kidney. Thus, the tissue distribution of regucalcin in rats was specific in the liver. The concentration of regucalcin in the liver was altered with increasing age of rats; liver regucalcin level linearly increased during 5 weeks old after birth of male rats, and then began to decrease gradually. The results coincided with the previous observation of Northern blot analyses by using liver regucalcin cDNA as a probe. The present finding clearly demonstrates that regucalcin is specifically synthesized in the liver of rats.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Role of regucalcin in calcium signaling

Regucalcin was discovered in 1978 as a calcium-binding protein that does not contain EF-hand motif of Ca(2+)-binding domain [M. Yamaguchi and T. Yamamoto, Chem. Pharm. Bull. 26 1915-1918 (1978)]. In recent years, regucalcin has been demonstrated to play an important role as a regulatory protein in Ca2+ signaling in rat liver and kidney cells. The organization of the rat regucalcin gene consists of seven exons and six introns. The mRNA is mainly present in liver and kidney with a size of 1.8 kb. Hepatic regucalcin mRNA expression has been shown to be stimulated by various factors including calcium, calcitonin, insulin, and estrogen in rats. The mRNA is also expressed in hepatoma cells (Morris hepatoma, HepG2, and rat hepatoma H4-II-E cells). Regucalcin plays a role in the maintenance of intracellular Ca2+ homeostasis due to activating Ca2+ pump enzymes in the plasma membrane (basolateral membrane) and microsomes of liver and renal cortex cells. Moreover, regucalcin has an inhibitory effect on the activation of Ca2+/calmodulin-dependent enzymes and protein kinase C. Also, regucalcin has been demonstrated to regulate nuclear function in liver cells; it can inhibit Ca(2+)-activated DNA fragmentation, DNA and RNA synthesis, protein kinase and protein phosphatase activities in the nuclei. Such an effect is also seen in the nuclei of regenerating rat liver. Regucalcin may play a physiological role in the control for overexpression of proliferative cells. Regucalcin has been proposed to be an important regulatory protein in Ca2+ signaling system, and it plays a multifunctional role in liver and kidney cells.     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

Phagostimulation of the mediterranean fruit fly, Ceratitis capitata by ribonucleotides and related compounds

Females of the medfly, Ceratitis capitata, prefer sucrose solutions containing ribonucleotides to sucrose solutions without them. The order of preference for the nucleotides was: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP=2 & 3GMP=RP>3AMP.2AMP, guanosine, inosine, adenine and 5TMP produced no significant stimulation. Females sterilized by irradiation showed reduced attraction to 5GMP as compared to non-irradiated females.Optimal molecular configuration for phagostimulation includes: phosphorylation at the 5 position of the ribose, free hydroxyl groups at 2 and 3 on the ribose, and an NH₂ group at the 2 position of the aromatic ring of purine.It is proposed that the 5GMP in yeast hydrolyzate can be used as a measure of the suitability of the hydrolyzate as a bait.

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Résumé La femelle de la mouche méditerranéenne des fruits, Ceratitis capitata, préfère les solutions de sucrose contenant des ribonucléotides aux simples solutions de sucrose. Lórdre de préférence pour les nucléotides est le suivant: 5GMP>GTP>5CMP>5IMP >dGMP>5UMP>5AMP>5XMP=ATP =2 & 3GMP=RP>3AMP.Le 2AMP, la guanosine, l'inosine, l'adénine et le 5TMP provoquent une stimulation significative. Les femelles montrent aprés stérilisation par irradiation une attirance réduite pour le 5GMP par comparaison avec les femelles non-irradiées.La configuration moléculaire optimale pour la phagostimulation comprend: la phosphorylation en position 5 du ribose; des groupes hydroxyles libres en 2 et 3 sur le ribose; et un groupe NH₂ en position 2 sur le noyau aromatique.Nous proposons que le 5GMP dans l'hydrolysat de levure puisse être utilisé pour mesurer la capacité de l'hydrolysat comme appât.

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Abbreviations 5AMP Adenosine 5-monophosphate - 3AMP Adenosine 3-monophosphate - 2AMP Adenosine 2-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - ADP Adenosine 5-diphosphate - ATP Adenosine 5-triphosphate - 5GMP Guanosine 5-monophosphate - 2GMP Guanosine 2-monophosphate - 3GMP Guanosine 3-monophosphate - dGMP 2-deoxyguanosine 5-monophosphate - GDP Guanosine 5-diphosphate - GTP Guanosine 5-triphosphate - 5IMP Inosine 5-monophosphate - IDP Inosine 5-diphosphate - ITP Inosine 5-triphosphate - 5XMP Xanthosine 5-monophosphate - 5CMP Cytidine 5-monophosphate - dCMP 2 deoxycytidine 5-monophosphate - CTP Cytidine 5-triphosphate - 5UMP Uridine 5-monophosphate - 5TMP Thymidine 5-monophosphate - RP Ribose 5 monophosphate