Effect of 17β-estradiol on cells stained for FSHβ and/or LHβ in the dog pituitary gland

Summary The effect of long-term treatment (52 weeks) with high doses of 17-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSH, canine GH, canine PRL and porcine ACTH¹. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LH or FSH, most cells were reactive to both antisera. The cells stained for FSH were less numerous than those shown to contain LH. TSH, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LH was observed. On the other hand, using the antiserum to FSH, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSH. However, a few cells were also shown to react solely with the antiserum to TSH. These cells, which seem to contain both TSH and FSH, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Mrs. K. Oertel for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Enzyme-catalyzed synthesis of alkyl β-D-glycosides with crude homogenate ofSulfolobus solfataricus

Summary Crude homogenate of thermophilic archaebacteriumSulfolobus solfataricus, possessing a -glycosidase, has been used to synthesize different alkyl -D-glycosides starting from phenyl -D-glucoside, phenyl -D-galactoside and lactose as carbohydrate donors. High product yield (95% with respect to the carbohydrate donor) of octyl -D-glucoside has been obtained in a two-phase system containing 5% of water. The enantioselection for the galactosyl transfer to the secondary hydroxyl group of propane-1,2-diol is higher than that found using -galactosidase fromE. coli.     

The isolation,characterization and sequence of two divergent β-tubulin genes from soybean (Glycine max L.)

Two divergent -tubulin genes (designated S-1 and S-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii -tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different -tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of -tubulin genes (thus far undetected) exist in the soybean genome. The S-1 and S-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode -tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with -tubulins from several organisms showed that they are most homologous to Chlamydomonas -tubulin (85–87%), with lesser degrees of homology to -tubulins of vertebrate species (79–83%), Trypanosoma brucei (80–81%) and Saccharomyces cerevisiae (66–68%). The amino acid sequences of S-1 and S-2 are as divergent from each other as they are from the Chlamydomonas -tubulin. The amino acids at the diverged positions in S-2 are nearly all conservative substitutions while in S-1, 18 of the 69 substitutions were non-conservative. Both soybean -tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas -tubulin genes. Codon usage in the two soybean -tubulins is remarkably similar (D ²=0.87), but differs from codon usage in other soybean genes.     

Über den histochemischen und mikrochemischen Nachweis der β-Galactosidase mit 1-Naphthyl-β-galactopyranosid

Zusammenfassung Es wird ein simultanes Azokupplungsverfahren zur intrazellulären Darstellung der sauren (Hetero-) und neutralen -Galactosidase (Lactase) in verschiedenen Organen von Ratte, Maus und Meerschweinchen beschrieben.Das Inkubationsmedium enthält 4,5–9mg 1-Naphthyl--galactopyranosid (gelöst in 0,4ml NN-Dimethylformamid) und 0,5–0,8ml 2% Hexazonium-p-rosanilin in 9 ml 0,1 M Citrat-Puffer, pH 5 (Hetero--galactosidase) oder 5,5 (Lactase).Unter allen Organen reagiert die saure -Galactosidase am kräftigsten in den Lysosomen von Nebenhoden, Niere, Nebenniere, Schilddrüse, Glandula präputialis und inguinalis, Milz, Colon und Plexus chorioideus; die neutrale -Galactosidase kommt in mittlerer Aktivität nur im intestinalen Stäbchensaum vor.Die intralysosomale Darstellung der löslichen Hetero--galactosidase erfordert Blockfixation in Glutaraldehyd; die Lactase kann an frischen oder gefriergetrockneten Schnitten untersucht werden. Im proximalen Tubulus der Rattenniere wird die saure -Galactosidase durch Formol unabhängig von der Konzentration des Fixans verglichen mit Glutaraldehyd stärker gehemmt. Spätestens 10 min nach Beginn der Fixation hat das Enzym seine Basisaktivität erreicht. Spülen in hypertoner Zuckerlösung macht die Inhibition der Hetero--galactosidase teilweise rückgängig.Die mit dem Azokupplungs- und Indigogen-Verfahren gewonnenen Befunde sind weitgehend identisch.

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On the histochemical and microchemical demonstration of -galactosidase by means of 1-naphthyl--galactopyranoside

Summary A simultaneous azo coupling method for the intracellular demonstration of acid (hetero-) and neutral -galactosidase (lactase) in various organs of rats, mice and guinea-pigs is described.The recommended incubation medium consists of 4.5–9 mg 1-naphthyl--galactopyranoside (dissolved in 0.4 ml NN-dimethylformamide) and 0.5–0.8 ml 2% hexazonium-p-rosaniline in 9 ml 0.1 M citrate buffer, pH 5.0 (hetero--galactosidase) or 5.5 (lactase).Among all organs investigated the strongest acid -galactosidase reaction regularly occurs in the lysosomes of the epididymis, kidney, adrenal, thyroid, preputial and inguinal gland, spleen, colon and chorioid plexus; the neutral -galactosidase can only be detected in the intestinal brush border exhibiting a moderate activity.Because hetero--galactosidase is a highly soluble enzyme bloc-fixation using glutaraldehyde becomes necessary to achieve a precise intralysosomal localization; for the demonstration of lactase fresh or freeze-dried cryostat sections are suitable. —In the proximal tubule of the rat kidney independent of their concentration the inhibition of acid -galactosidase following treatment with formol surpasses that of glutaraldehyde. Within the first ten minutes of fixation the enzyme reaches its basis activity. The recovery rate of renal hetero--galactosidase considerably increases in the course of washing in hypertonic sugar solution.In comparison with the indigogenic technique nearly identical results can be obtained with the azo coupling procedure.

     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Simultaneous overproduction of extracellular endoglucanase and intracellular β-glucosidase by genetically engineered Bacillus subtilis

Summary Simultaneous overproduction of intracellular -glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the -glucosidase-endoglucanase gene(E) or the endoglucanase--glucosidase gene(E) under the control of a strong engineered promoter, BJ27U88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the -glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Significance of bacterial ectoenzymes in aquatic environments

The report presents studies on temporal and spatial variations of kinetics (Vmax and Km) of bacterial ectoenzyme activity (-glucosidase - Glc, leucine aminopeptidase - Leu-amp) in the naturally eutrophic Plusee. Glc and Leu-amp activity were positively correlated with the flux of polymeric materials (polysaccharides, proteins) in the lake. Glc activity was low when algal populations grew actively, but during the algal bloom breakdown Glc activity increased rapidly. Leu-amp displayed the highest rates of activity in the epilimnion and was tightly coupled to bacterial production. The synthesis of studied ectoenzymes was under control of a repression/derepression mechanism. The significance of ectoenzymes for the transformation and bacterial utilization of organic matter, and their role in the microbial loop in aquatic environments is discussed.     

Über den histochemischen Nachweis der β-Glueosidase mit 1-Naphthyl-β-glucopyranosid

Zusammenfassung Es wird eine einfache simultane Azokupplungsmethode zur Darstellung der -Glucosidase im Dünndarm verschiedener Säuger beschrieben und mit anderen histochemischen Verfahren zum Nachweis dieses Enzyms verglichen. Eine intrazelluläre Lokalisation der -Glucosidase ermöglichen nur die Indigogen-und die hier angegebene Technik, nicht dagegen die bisherigen Azofarbstoffmethoden mit 6-Br-2-Naphthyl--glucopyranosid als Substrat und p-Rosanilin zur Simultanoder Fast Blue B zur Postkupplung.Das Inkubationsmedium des neuen Verfahrens enthält 4,5–9,0 mg 1-Naphthyl--glucopyranosid (gelöst in 0,4 ml Dimethylformamid) und 0,4–0.8 ml 2% hexazotiertes p-Rosanilin in 9,0 ml 0,1 M Citronensäure-Phosphat-Puffer, pH 5,5. —Mikrochemische Messungen mit dem gleichen Substrat zeigen, daß die -Glucosidase durch p-Rosanilin in ähnlichem Ausmaß wie durch Ferricyanid im Indigogen-Medium gehemmt wird.Wegen der fraglichen Verwandtschaft von -Glucosidase und neutraler -Galactosidase wurde dieses Enzym mit obigem Ansatz und 1-Naphthyl--galactopyranosid als Substrat untersucht.

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On the histochemical demonstration of -glucosidase with 1-naphthyl--glucopyranoside

Summary A simple simultaneous azo coupling method for the demonstration of -glucosidase in the small intestine of various mammals is described and compared with other histochemical techniques for this enzyme. Strong evidence occurs that a correct intracellular localization of -glucosidase can only be obtained by means of the indigogenic and the assay presented here: the azo dye methods published so far with 6-Br-2-naphthyl--glucopyranoside as substrate and p-rosaniline for simultaneous or Fast Blue B for postcoupling are not able to reflect the true binding sites of intestinal--glucosidase.The recommended incubation medium consists of 4.5–9.0 mg 1-naphthyl--glucopyranoside (dissolved in 0.4 ml NN-dimethyl formamide) and 0.6–0.8 ml 2% hexazonium-p-rosaniline in 9.0 ml 0.1 M citric acid-phosphate buffer, pH 5.5.— Microchemical measurements using the same substrate show that p-rosaniline inhibits -glucosidase to a similar extent as ferricyanide in the indigogenic medium.Because of the presumed relationship between -glucosidase and neutral -galactosidase the latter enzyme has been demonstrated with the above mentioned assay replacing the 1-naphthyl--glucoside by the corresponding -galactopyranoside.The strongest -glucosidase and -galactosidase activity can regularly be observed in the jejunum of rats, mice and guinea-pigs where both enzymes are localized in the brush border region of the enterocytes. In comparison with -galactosidase the -glucosidase reaction is always more intensive and the azo dye production in the microvillous zone of suckling rats and guinea-pigs is far higher than in the intestine of adult animals. Furthermore both enzymes react in a similar way to inhibitors, experiments (thirst, hunger) and pregnancy and do not split naphthol AS BI -glucopyranoside respectively -galactopyranoside.Bloc fixation in formol-calcium and especially in glutaraldehyde improves the localization of the azo dye considerably; but microchemistry reveals that aldehyde fixation supresses the -glucosidase to ca. 50%. The basis activity of the enzyme following pretreatment with formol is reached within the first minute of fixation.

     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Developmental changes in neutral glycosphingolipids of mouse placenta

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive spot migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of¹²⁵I labelledHelix pomatia agglutinin (-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.Abbreviations asialo-GM₁ Gal 3GalNAc4Gal4Glc1Cer - BCIP 5-bromo-4-chloro-3-indolylphosphate - DHC lactosylceramide, Gal4Glc1Cer - Forssman antigen GalNAc3GalNAc3Gal4Gal4Glc1Cer - globoside GalNAc3Gal4Gal4Glc1Cer - GSL glycosphingolipids - HPA Helix pomatia agglutinin - HPTLC high performance thin layer chromatography - MHC galactosylceramide, Gal1Cer - MHC glucosylceramide, Glc1Cer - PBS phosphate-buffered saline - PNA peanut agglutinin - PVP poly(vinylpyrrolidone), mol. wt 40 000 - SBA soybean agglutinin - THC trihexosylceramide, Gal4Gal4Glc1Cer. To whom correspondence should be addressed.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Developmental aspects of the innervation of skeletal muscle fibers in the chick embryo

Summary The M. complexus in the chick, commonly called the hatching muscle, undergoes conspicuous growth during the latter stages of embryonic development. Myogenesis of this muscle was compared to that of M. biceps femoris with regard to development of types of muscle fiber and their innervation. In both muscles fibers are of relatively uniform size and show little growth in diameter between 12 days of development and hatching; fibers develop continuously and display a wide range of diameters at all stages.Initial thickenings on the sarcolemma of fibers where axons are closely approximate were first observed at 10 days of development in both muscles. In both muscles fibers are innervated prior to fibers. Terminal axon networks bridge intercellular spaces and contact fibers in different myogenic clusters, fibers that develop on the surface membrane of fibers exhibit focal thickenings of the membrane and some cell projections that are directed toward axon- fiber contacts. These changes occurred only in fibers of M. complexus.At 14 days of embryogenesis, the processes of synaptogenesis and of myelin formation are less advanced in M. biceps femoris than in M. complexus. At this stage a fibers were observed to be innervated in M. complexus, but not yet in M. biceps femoris. Each fiber was observed to be encircled by several preterminal axons.It is concluded that the earlier development of M. complexus is correlated with an equally early development of nerve-muscle interactions.This work was supported in part by a grant from the Muscular Dystrophy Association of America, Inc.Postdoctoral Fellow of the Muscular Dystrophy Association I would like to thank Professor Dr. H. Tamate for his valuable advise. I am also grateful to Dr. L. Doerr, H. Stokes and Judi K. Lund for their advice and skilled technical assistance     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

Elimination of the disulphide bond alters the conformation of mature lipo-β-lactamase in yeast

Summary When expressed in Saccharomyces cerevisiae the precursor of the hybrid prokaryotic protein lipo--lactamase is accumulated in reduced form whereas the majority of the mature form contains an intra molecular disulphide bond (oxidized form). We have previously shown that mature mutant lipo--lactamases in which the cysteine residue 131 was changed to either tyrosine or threonine lack the capacity to form a disulphide bond but are nevertheless processed and secreted efficiently in yeast. Here we show that these mutant mature lipo--lactamases, in yeast cell extracts, exhibit a tenfold lower -lactamase-specific activity than the wild-type protein and that the mature mutant proteins are more susceptible to trypsin digestion. Thus, elimination of the disulphide bond alters the conformation of mature lipo--lactamase in yeast.Correspondence to: O. Pines     

Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Ein fluoreszenzmikroskopischer Nachweis von β-Glucosidase in Wurzeln vonCicer arietinum (L.)

Zusammenfassung Die Lokalisierung von -Glucosidasen in Wurzeln der Kichererbse [Cicer arietinum (L.)] wurde in einem einstufigen fluoreszenz-optischen Verfahren mit 4-Methyl-umbelliferyl-D-glucosid untersucht. Am Beispiel der Kichererbse wird gezeigt, daß in polyphenolhaltigen Pflanzen herkömmliche Azokupplungsverfahren zur Glucosidaselokalisierung nicht anwendbar sind. Die mit der neuen Methode erhaltenen Ergebnisse decken sich im wesentlichen mit denen aus vergleichbaren Untersuchungen und zeigen, daß -Glucosidasen nicht vorhanden sind. Aryl--Glucosidasen wurden vorwiegend im äußeren Cortexbereich gefunden und nehmen mengenmäßig zur Wurzelspitze hin zu.

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Localization of -glucosidase in roots ofCicer arietinum (L.) by a fluorescence technique

Summary The localization of -glucosidases in roots of garbanzo beans [Cicer arietinum (L.)] has been investigated by a one-step fluorescence optical procedure using 4-methylumbelliferyl--D-glucoside. It is shown that the well known azo-coupling test for the localization of glucosidases cannot be applied in polyphenol containing plants. The results obtained with the new method are comparable with those described in other investigations and further show that -glucosidases are absent from the root tissues investigated. The aryl--glucosidases were mainly detected in the outer region of the cortex and quantitatively increase towards the root tip.