Cellular localization of glycoside hydrolases inBacteroides fragilis

The localizations of six glycosidases produced byBacteroides fragilis—-glucosidase, -glucosidase, -galactosidase, -galactosidase, -N-acetylglucosaminidase, and -l-fucosidase—were studied. Cell fractions and cell extracts were obtained by Triton X-100 release, by disruption by freeze-pressing and sonication, and by osmotic release. Isoelectric focusing of a cytoplasmic and of a Triton X-100 extract of the cell wall fraction was performed and revealed differences in the relative distribution of differently charged forms of -N-acetylglucosaminidase. -Galactosidase and alkaline phosphatase were used as cytoplasmic and periplasmic markers, respectively. It is concluded that inB. fragilis -glucosidase is periplasmic, -l-fucosidase and -galactosidase are cytoplasmic, and -n-acetylglucosaminidase is cell associated and bound to the cell envelope by hydrophobic interactions. -Glucosidase and -galactosidase are localized cytoplasmically and/or located in the cell envelope.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Secretion of lysosomal enzymes by human placental villi in vitro

Summary Chorionic villi from first trimester and term human placentas have been incubated in vitro and shown to release the lysosomal enzymes, -hexosaminidase, -glucosidase and -gluctlronidase. There was negligible release of the cytoplasmic enzyme, lactate dehydrogenase, under the same conditions. The first trimester villi released proportionally more of their lysosomal enzyme content than did the term villi. Extracellular levels of -hexosaminidase were raised and those of -glucosidase and, -glucuronidase were lowered when tissue was incubated with 1 M colchicine, suggesting that microtubules are involved in the control of lysosomal enzyme release from placental villi.     

The histochemical demonstration of five lysosomal enzymes in the pars distalis of the amphibian pituitary

Summary The distribution of five lysosomal enzymes (acid phosphatase, -glucosaminidase, -glucuronidase, sulphatase and E 600-resistant esterases) was studied in the pars distalis of the urodelian pituitary. Naphthol-AS-compounds coupled with hexazonium-pararosanilin yielded particularly good localization. The typical lysosomal picture and a basically similar distribution were obtained throughout. The reaction product was mainly found in the globules of the globular basophils (-cells) reported as gonatropin producing cells by several authors. Some speculations on the possible function of the high amount of the lysosomal enzymes in the globular basophils are made.Dedicated to Professor W. Bargmann on his 60th birthday.This work was partly supported by a grant from Deutscher Akademischer Austauschdienst, Bad Godesberg.     

Histochemical studies on the distribution of β-glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of the rat

Summary The present study compares the distribution of -glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of rat. In young cells there are indications of cyclic activity of these enzymes, i.e., in some stages there are perinuclear concentrations of the enzymes, at other times -glucuronidase and succinic dehydrogenase are uniformly distributed throughout the cytoplasm. These stages have been discussed with the identical distribution of mitochondria. However, in old spinal ganglion cells both -glucuronidase and succinic dehydrogenase become mainly concentrated in the pigment areas, suggesting thereby their possible role in the production of pigment, through the medium of the mitochondria.     

Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, 5-3-hydroxysteroid dehydrogenase (5-3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD). The activities of acid phosphatase, alkaline phosphatase and -glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular 5-3-HSD activity was higher in transgenic than in normal mice, while testicular 17-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of -glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.     

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.     

Verteilung und Aktivität hydrolytischer Enzyme in Hypophysenadenomen

Zusammenfassung An 17 Hypophysenadenomen [12 Chromophobe (endokrin-inaktive), 5 Mischtypadenome (endokrin-aktive)] wurde das Verhalten und die Aktivität der hydrolytischen Enzyme -Galaktosidase, -Glucuronidase, -Glykosidase und Arylsulfatase mit histochemischen Methoden geprüft.Die Mischtypadenome zeigen insgesamt eine höhere Aktivität als die Chromophoben. Dabei reagieren -Galaktosidase und -Glykosidase am stärksten, -Glucuronidase etwas schwächer, während Arylsulfatase die niedrigste Aktivität zeigt. Die Befunde werden mit anderen enzymhistochemischen Untersuchungen an Hypophysenadenomen und tierexperimentellen Ergebnissen verglichen. Daraus folgt, daß wahrscheinlich zwischen der Aktivität der untersuchten lysosomalen Enzyme und der endokrinen Aktivität ein Zusammenhang besteht.

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Summary Behaviour and activity of the hydrolytic enzymes -galactosidase, -glucuronidase, -glycosidase and arylsulphatase are tested in 17 adenomas of the hypophysis (12 chromophobic, endocrine-inactive; 5 mixed cell adenomas, endocrine-active).Mixed cell adenomas show an altogether higher activity than chromophobic adenomas. -galactosidase and -glycosidase show the highest, -glucuronidase a slightly lower, and arylsulphatase the lowest activity. The findings are compared with other enzymhistochemical methods and results from animal experiments. The results of this comparison indicate that there is a correlation between the endocrine activity of the lysosomal enzymes in question.

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Der Deutschen Forschungsgemeinschaft danken wir für ihre Unterstützung.

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Fräulein Renate Kott, Fräulein Marianne Lehnen und Fräulein Edith Klasmeier danke ich für ihre technische Unterstützung.     

Sterols of male and female compound inflorescences of Zea mays L.

A comparison has been made between the sterols of male and female inflorescences and of pollen from Zea mays. The female inflorescence was shown to contain cholesterol, 24-methylcholesterol, 24-ethyl-5,22-cholestadien-3-ol, 24-ethylcholesterol and (28Z)-24-ethylidenecholesterol. Themale inflorescence contained the same five compounds together with 24-methylenecholesterol. Pollen contained 24-methylenecholesterol as its main sterol together with lesser amounts of cholesterol, 24-ethylcholesterol, (28Z)-24-ethylidenecholesterol, 24-methylene-5-cholest-7-en-3-ol and 4-methyl-24-methylene-5-cholest-7-en-3-ol.     

Localization of lysosomal antigens in activated T-lymphocytes

Summary The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel, electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme -glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that -glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density.Fractionation of homogenates from lymphocytes on Percoll gradients revealed that -glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of -glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.     

Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

Summary The dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney -glucuronidase and acid phosphatase. Use was made of the renal -glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of marker enzymes and by observations with the electron microscope. In all subcellular fractions, -glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal -glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal -glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble -glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal -glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these -glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and -glucuronidase as enzyme markers for lysosomes in mouse kidney.Aided in part by Research Grant, P-106, of the American Cancer Society, Inc., New York, and by U.S.P.H.S. Grant CA-07538 and by a Research Career Award, CA-K6-18453 to William H. Fishman.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Molecular analysis of methane monooxygenase from Methylococcus capsulatus (Bath)

Methane monooxygenase (MMO) is the enzyme responsible for the conversion of methane to methanol in methanotrophic bacteria. In addition, this enzyme complex oxidizes a wide range of aliphatic and aromatic compounds in a number of potentially useful biotransformations. In this study, we have used biochemical data obtained from purification and characterization of the soluble MMO from Methylococcus capsulatus (Bath), to identify structural genes encoding this enzyme by oligonucleotide probing. The genes encoding the and subunits of MMO were found to be chromosomally located and were linked in this organism. We report here on the analysis of a recombinant plasmid containing 12 kilobases of Methylococcus DNA and provide the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex. DNA sequence analysis suggests that the primary structures of the and subunit of MMO are completely novel and the complete sequence of these genes is presented.     

Further observations on cell-wall formation around isolated protoplasts of tobacco and tomato

Freeze-etch observations of protoplasts isolated from tobacco (Nicotiana tabacum L.) mesophyll tissue and tomato (Lycopersicum esculentum Mill.) fruit locule tissue are described which clarify earlier observations (Burgess, J., Fleming, E.N., Planta 131, 173–178, 1976; Planta 133, 267–273, 1977), obtained using scanning electron microscopy. of fibres associated with projections from these cell surfaces. It is demonstrated (1) that the fibres consist of bundles of small numbers of microfibrils which have become artifactually thickened by the deposition of coating materials, and (2) that the apparent association between fibres and projections results from microfibrils being lifted preferentially from protoplast surfaces in regions rich in projections (plasmalemmasomes). With the higher resolution available using freeze-etching it can be demonstrated that microfibril deposition does not occur in discontinuous zones on these protoplast surfaces. Globules associated with microfibril termini in radish (Raphanus sativus L.) roots are illustrated and it is proposed that turgor pressure differences between isolated protoplasts and intact tissue may account for the absence of similar globules from isolated protoplast surfaces.     

Purification of β-glucuronidase and structural assessment of the carbohydrate chains by lectin affinity immunoelectrophoresis

The purification of rat liver -glucuronidase from a lysosomal fraction by methods including affinity chromatography, chromatofocusing and preparative PAGE steps is described. Molecular weights of 300 000 and 150 000 were estimated by two dimensional gradient PAGE/immunoelectrophoresis of the lysosomal extract. Isoelectrofocusing in agarose gel followed by immunoelectrophoresis in the second dimension revealed the presence of at least five maxima in the range pH 4.3–7.4.The structural assessment of the carbohydrate chains of lysosomal and microsomal -glucuronidase was performed by lectin affinity immunoelectrophoresis. Reaction with Concanavalin A indicated the presence of bi-antennary complex, oligomannosidic and hybrid type structures, whereas the absence of tri- and tetra-antennary complex type structures was deduced from the lack of interaction with phytohemagglutinin-L. The reaction withLens culinaris agglutinin, Pisum sativum agglutinin andLotus tetragonolobus lectin revealed that part of the glycans contained a fucose (1-6)-linked to theN-acetylglucosamine attached to asparagine. The presence of terminal (1-4)-galactose residues was detected withRicinus communis agglutinin I.     

Histochemistry of some acid hydrolases in striated muscles of the rat

Summary The distribution of acid phosphatase, -n-acetylglucosaminidase, -glucuronidase, and acid -galactosidase was studied in mm. extensor digitorum longus, soleus, and diaphragm of rats. Using the technic of semipermeable membranes activities of these enzymes were demonstrated beside cells of the interstitial tissue in muscle fibers themselves as well. Acid phosphatase displayed the highest activity which appeared in many small dots dispersed in the fiber. The activity of acid phosphatase was about 1.2 x higher in the m. soleus than in the m. extensor digitorum longus. In the latter muscle a somewhat higher activity was often found in muscle fibers displaying a higher staining for NADH tetrazolium reductase. The activity of -n-acetylglucosaminidase was slightly lower, that of -glucuronidase very weak but still discernible. The activity of acid -galactosidase was not ascertained in the majority of fibers. The ratio of activities measured in an area of the same size in cells of the interstitial tissue and in muscle fibers amounted in average to 2.6: 1 in the case of acid phosphatase, 2.5:1 in the case of -n-acetylglucosaminidase, 5.7: 1 in the case of -glucuronidase, and 44.3:1 in the case of acid -galactosidase. The importance of the histochemical technic in studies concerned with acid hydrolases in striated muscle fibers in normal and pathological conditions is pointed out.