Xanthindehydrogenase in Organen vonDrosophila melanogaster

Summary High activity of the enzyme, xanthindehydrogenase, was detected in larval fat-body of wild typeDrosophila melanogaster. The Malpighian tubules of both larvae and adults exhibit activity, too, though rather little. No activity was detected in larval testes, while probably insignificant traces were found in adult testes. The results are discussed with regard to non-autonomy of mutants deficient in the enzyme.

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Ausgeführt mit Unterstützung des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung.     

Vesicular inclusions in the nuclei of epithelial cells of Malpighian tubes of the hemipteran,Panstrongylus megistus

Summary Vesicular inclusions observed in Malpighian tubes of hemipterans have been associated with a virus-like infection rather than a lysosomal-type activity, which is the case of the identical cytoplasmic structures.This study was supported by a grant (75/907) of the Fundação de Amparo à Pesquisa do Estado de São Paulo. The authors are indebted to Dr E. O. Rocha-e-Silva for generously providing them with the insect specimens.     

On the distribution of plasma L-asparaginase

Summary The guinea-pig,Cavia porcellus, is unusual in possessing plasma L-asparaginase, an enzyme with anti-tumor activity. 21 additional species have been examined as to the presence of this enzyme: the results confirm and extend its remarkably limited species distribution.This work was supported in part by grant GM/CA 25378 from the National Institute of General Medical Sciences, National Institutes of Health.We are grateful to dr Hanna Hensch, Philadelphia Zoological Garden, and Ms Joan Whitla and Dr Richard J. Montali, National Zoological Park/Smithsonian Institution, for samples of frozen serum.To whom correspondence should be addressed.     

The effect of temperature on the ouabain-sensitivity of Na+−K+-activated ATPase and fluid secretion by the Malpighian tubules ofLocusta

Summary Temperature markedly affected the ouabain-sensitivity of both the Na⁺–K⁺-activated ATPase activity and the secretion of fluid by the Malpighian tubules ofLocusta. Varying the K⁺ concentration in the bathing medium did not affect the ouabain-sensitivity of the fluid secretory process.     

Autoradiographic study of the penetration of non-histone chromatin proteins into differentiating cells

Summary Attention has previously been drawn to a specific effect of NHCP on embryonicPleurodeles cell differentiation. With a modified NHCP labelling technique, autoradiography has revealed a cytoplasmic concentration of labelled NHCP and has not revealed any difference between homospecific and heterospecific NHCP penetration.This work has been supported financially by research grants from the C.N.R.S. (ERA No. 327 and ATP No. 4310 on the cellular differentiation), and INSERM (No. 711019).The authors wish to extend their thands to Dr.Mazarguil who kindly initiated us in the techniques of enzyme binding. Many thanks are also due to Mr.Hawks for most valuable help in correcting the English.     

Synthesis of a ubiquitously present new HSP60 family protein is enhanced by heat shock only in the Malpighian tubules ofDrosophila

A homologue of the chaperonin protein of the HSP60 family has not been shown so far inDrosophila. Using an antibody specific to HSP60 family protein in Western blotting and immunocytochemistry, we showed that a 64-kDa polypeptide, homologous to the HSP60, is constitutively present in all tissues ofDrosophila melanogaster throughout the life cycle from the freshly laid egg to all embryonic, larval and adult stages. A 64-kDa polypeptide reacting with the same antibody in Western blots is present in all species ofDrosophila examined. Using Western blotting in conjunction with³⁵S-methionine labeling of newly synthesized proteins and immuno-precipitation of the labeled proteins with HSP60-specific antibody, it was shown that synthesis of the 64-kDa homologue of HSP60 is appreciably increased by heat shock only in the Malpighian tubules, which are already known to lack the common HSPs.     

Invertase activity in the gut of 6th instar larvae ofSpodoptera mauritia Boisd. (Noctuidae,Lepidoptera)

Summary Invertase activity has been studied in the fore-, mid- and hindgut of the 6th instar larva ofSpodoptera mauritia. The highest activity was in the midgut except during the early hours of the larval period when the foregut showed comparatively increased activity. The hindgut invertase activity may be from the voiding of enzyme along with the undigested food.Acknowledgments. The authors wish to thank the Department of Zoology, for providing the facilities for this work. The senior author gratefully acknowledges the junior research fellowship from the University of Calicut.     

Water in enzyme reactions: biophysical aspects of hydration-dehydration processes

Water has been recognized as one of the major structuring factors in biological macromolecules. Indeed, water clusters influence many aspects of biological function, and the water-protein interaction has long been recognized as a major determinant of chain folding, conformational stability, internal dynamics, binding specificity and catalysis. I discuss here several themes arising from recent progress in understanding structural aspects of ‘direct’ and ‘indirect’ ligands in terms of enzyme-substrate interactions, and the role of water bridges in enzyme catalysis. The review also attempts to illuminate issues relating to efficiency, through solvent interactions associated with enzymic specificity, and versatility. Over the years, carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) has played a significant role in the continuing delineation of principles underlying the role of water in enzyme reactions. As a result of its pronounced catalytic power and robust constitution CA was transformed into a veritable ‘laboratory’ in which active site mechanisms were rigorously tested and explored.     

Comparative study of oxalate oxidase in three genotypes ofSorghum vulgare

Summary The presence of an oxalate oxidase (EC 1.2.3.4) has been demonstrated in 15,000×g supernatants prepared from 10-day-old seedlings of three genotypes ofSorghum vulgare: grain sorghum hybrid (CSH-5), grain-cum-forage sorghum (PC-6) and forage sorghum (PC-1). The specific activity of the enzyme in the different tissues of seedlings was found to be present in the order leaves > stems > roots in PC-6 and PC-1, but this order was reversed in CSH-5. A comparison of the different properties of the leaf enzyme of these three genotypes of sorghum revealed that the enzyme has maximum activity in the acidic pH range from 4.0 to 5.0 and in the temperature range from 37°C to 40°C. The enzyme was stimulated by Cu²⁺ and Fe²⁺. The rate of H₂O₂ formation in the enzyme reaction was linear up to 5 min and was stoichiometrically related to oxalate consumption. The enzyme is unaffected by Na⁺ at physiological concentration (0.15 M). The superiority of this enzyme over moss and other plant enzymes for enzymic determination of urinary oxalate is discussed.     

Unique evolution of Bivalvia arginine kinases

The clams Pseudocardium, Solen, Corbicula and Ensis possess a unique form of arginine kinase (AK) with a molecular mass of 80 kDa and an unusual two-domain structure, a result of gene duplication and subsequent fusion. These AKs also lack two functionally important amino acid residues, Asp⁶² and Arg¹⁹³, which are strictly conserved in other 40-kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. However, these AKs show higher enzyme activity. The cDNA-derived amino acid sequences of 40-kDa AKs from the blood clam Scapharca broughtonii and the oyster Crassostrea gigas were determined. While Asp⁶² and Arg¹⁹³ are conserved in Scapharca AK, these two key residues are replaced by Asn and Lys, respectively, in Crassostrea AK. The native enzyme from Crassostrea and both of the recombinant enzymes show an enzyme activity similar to that of two-domain clam AKs and at least twofold higher than that of other molluskan AKs. Although the replacement of Asp⁶² or Arg¹⁹³ by Gly in normal AK causes a considerable decrease in V_max (6–15% of wild-type enzyme) and a two- to threefold increase in K_m for arginine, the same replacement in Scapharca AK had no pronounced effect on enzyme activity. Together with the observation that bivalve AKs are phylogenetically distinct from other molluskan AKs, these results suggest that bivalve AKs have undergone a unique molecular evolution; the characteristic stabilizing function of residues 62 and 193 has been lost and, consequently, the enzyme shows higher activity than normal.Received 14 October 2003; accepted 1 November 2003     

Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5′-nucleotidase

Human erythrocyte pyrimidine 5′-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.Received 25 March 2005; received after revision 3 May 2005; accepted 13 May 2005     

Acetyl-coenzyme A synthetase (AMP forming)

Acetyl-coenzyme A synthetase (AMP forming; Acs) is an enzyme whose activity is central to the metabolism of prokaryotic and eukaryotic cells. The physiological role of this enzyme is to activate acetate to acetyl-coenzyme A (Ac-CoA). The importance of Acs has been recognized for decades, since it provides the cell the two-carbon metabolite used in many anabolic and energy generation processes. In the last decade researchers have learned how carefully the cell monitors the synthesis and activity of this enzyme. In eukaryotes and prokaryotes, complex regulatory systems control acs gene expression as a function carbon flux, with a second layer of regulation exerted posttranslationally by the NAD⁺/sirtuin-dependent protein acetylation/deacetylation system. Recent structural work provides snapshots of the dramatic conformational changes Acs undergoes during catalysis. Future work on the regulation of acs gene expression will expand our understanding of metabolic integration, while structure/function studies will reveal more details of the function of this splendid molecular machine.Received 4 December 2003; received after revision 2 March 2004; accepted 16 March 2004     

Juvenile hormone and reproduction in the cricket. II. Effect of rearing temperature on corpus allatum activity (in vitro) in adult females

Summary In the Mediterranean field cricket,Gryllus bimaculatus, reproduction is controlled by temperature and the corpus allatum (CA) hormone JH III. In CA of females reared at 24°12°C(168 h) (high reproduction rate) a first peak in JH III synthesis is reached about 4 days earlier than in those of 20°C females (low reproduction rate). Furthermore, in 20°C animals CA activity is low during the entire oviposition period, whereas at 24°12°C high CA activity is found during this period of adult life. The results indicate a stimulation of CA activity and reproduction by thermoperiods around a constant low temperature.Supported by the DFG (SFB 87 A 4).     

Circadian rhythm ofβ-glucuronidase

Summary The circadian rhythm of rat liver-glucuronidase has been studied in animals kept under highly standardized laboratory condition. A clear 24 h rhythm has been observed for this enzyme with a peak activity at 01.00 h and a trough at 13.00 h.The work was carried out in Institut für Anatomie I, Medical School, Hannover (Federal Republic of Germany). Supported by grants from Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 146 and VW Stiftung, Hannover A2 11.2000.     

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172. Received 7 December 1998; received after revision 15 March 1999; accepted 22 March 1999     

The mammalian NudC-like genes: a family with functions other than regulating nuclear distribution

Nuclear distribution gene C homolog (NudC) is a highly conserved gene. It has been identified in different species from fungi to mammals. The high degree of conservation, in special in the nudC domain, suggests that they are genes with essential functions. Most of the identified genes in the family have been implicated in cell division through the regulation of cytoplasmic dynein. As for mammalian genes, human NUDC has been implicated in the migration and proliferation of tumor cells and has therefore been considered a possible therapeutic target. There is evidence suggesting that mammalian NudC is also implicated in the regulation of the inflammatory response and in thrombopoiesis. The presence of these other functions not related to the interaction with molecular motors agrees with that these genes and their products are larger in size than their microbial orthologous, indicating that they have evolved to convey additional features.     

Peroxidase activity in the sponge,Iotrochota birotulata

Summary The spongeIotrochota birotulata contains a peroxidase which was partially characterized. This is the first report of a peroxidase in Porifera, originally thought to be devoid of such enzymatic activity.This study was supported by grant No. CA 21992 from the National Cancer Institute, DHEW.     

Friedrich Miescher Prize awardee lecture review. A conserved family of nuclear export receptors mediates the exit of messenger RNA to the cytoplasm

The distinguishing feature of eukaryotic cells is the segregation of RNA biogenesis and DNA replication in the nucleus, separate from the cytoplasmic machinery for protein synthesis. As a consequence, messenger RNAs (mRNAs) and all cytoplasmic RNAs from nuclear origin need to be transported from their site of synthesis in the nucleus to their final cytoplasmic destination. Nuclear export occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors, which shuttle between the nucleus and cytoplasm. The past years have seen great progress in the characterization of the mRNA export pathway and the identification of proteins involved in this process. A novel family of nuclear export receptors (the NXF family), distinct from the well-characterized family of importin β-like proteins, has been implicated in the export of mRNA to the cytoplasm. Received 23 January 2001; received after revision 12 April 2001; accepted 12 April 2001     

Chymotrypsin gene expression during the intermolt cycle in the shrimpPenaeus vannamei (Crustacea; Decapoda)

InPenaeus vannamei, chymotrypsin is present as two isoenzymes in the hepatopancreas. The enzyme has been localized in F-cells by immunocytochemistry using a specific antibody. By in situ hybridization, with a 510 pb cDNA probe encoding for the first 170 amino acids of the shrimp chymotrypsin, mRNA was localized in the same cells. Gene expression was followed during the intermolt cycle by measuring changes in specific activity in crude extracts, and by the estimation of mRNA levels by Northern blots using the same probe. The increase in specific activity in premolt is preceded in early premolt by an increase in the amount of chymotrypsin mRNA. A second increase is observed in postmolt, suggesting a different mode of regulation of gene expression.     

Le rôle de l'hétérosis dans la compétition entreebony et son allèle normal

Summary Whereas in homozygous state the geneebony noticeably diminishes the sexual activity of the males, it can on the contrary increase it in heterozygous state; but this heterotic effect depends in a large measure on the direction of the crossing, and also on the genic and cytoplasmic context. It depends moreover on the temperature.The role of this activity in the populations whereebony is made to compete with its normal allel is discussed briefly.