Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins

The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa subunits of F₁-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex. Both mitochondrial F₁ precursors were specifically processed by a soluble stromal extract from chloroplasts. However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract. The cleavage of the mitochondrial F₁ precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline. The cleavage site of the mitochondrial F₁ precursor by the chloroplast soluble extract appears to be located at the N-terminus.Abbreviations ATPase adenosine triphosphatase - Rieske FeS non-heme iron sulphur protein of the ubiquinol cytochrome c oxidoreductase complex - Rubisco ribulose 1,5-bisphosphate carboxygenase/oxygenase - RMSF phenylmethylsulphonylfluoride - EDTA ethylenediaminetetraacetic acid     

Localization of Filipin-Sterol Complexes in the Membranes of Beta vulgaris Roots and Spinacia oleracea Chloroplasts

Filipin was used as a cytochemical probe for membrane sterols in the root storage tissue of the red beet Beta vulgaris L. and the chloroplasts of Spinacia oleracea L. In unfixed beet tissue, filipin lysed the cells. Freeze-fracture replicas revealed that the filipin-sterol complexes were tightly aggregated in the plasma membrane, while in thin section the complexes corrugated the plasma membrane. If the cells were fixed with glutaraldehyde prior to the filipin treatment, the cell structure was preserved. Filipin-induced lesions were dispersed or clustered loosely in the plasma membrane. A few filipin-sterol complexes were observed in the tonoplast. In spinach chloroplasts, filipin-sterol complexes were limited to the outer membrane of the envelope and were not found in the inner membrane of the envelope or in the lamellar membranes. If the filipin-sterol complexes accurately mapped the distribution of membrane sterols, then sterol was located predominantly in the plasma membrane of the red beet and in the outer membrane of the chloroplast envelope. Furthermore, the sterol may be heterogenously distributed laterally in both these membranes.     

beta-Hydroxysterol distribution as determined by freeze-fracture cytochemistry

Summary Filipin a polyene antibiotic, fluoresces and forms 15–25 nm aggregates when combined with -hydroxysterols, rendering sterols detectable by fluorescence microscopy and by electron microscopy of thin sections and freeze-fracture replicas. We applied filipin in a glutaraldehyde fixative to tissue-cultured cells ofDrosophila melanogaster larvae, in which sterol concentration can be regulated. Since the number of filipin-sterol aggregates observed in membranes was found to be preportional to the amount of sterol experimentally inserted, utilizing filipin is a valid method for quantifying, as well as for mapping, sterol distribution in biological membranes. Other antibiotics may be similarly used for localizing some species of negatively charged phospholipids.In addition to cytochemical identification of specific lipids, rapid freezing and deep etching of unfixed, non-cryoprotected cells may permit us to examine membrane lipids in different physical states liquid-crystalline and gel. Combining these several techniques has resulted in new data concerning the disposition of lipids during the intimate juxtaposition of membranes preceding fusion. For example, in guinea-pig sperm, foci of closely apposed membranes are bereft of -hydroxysterols and intramembranous particles. Such regions of membrane sometimes exist in a crystalline state and may be rimmed by negatively charged phospholipids. As previously noted in other areas of cytochemistry, thein situ localization of specific substances provides information unobtainable by morphological or biochemical techniques alone.     

Die fluchtreaktionen der ErbsenlausAcyrthosiphon pisum ausgel?st durch Alarmpheromon und zus?tzliche reize

Zusammenfassung Der Duft von (E)--Farnesen veranlaßte in hohen Konzentrationen 84% ungeflügelter Erbsenläuse (Acyrthosiphon pisum (Harris)) innerhalb von 10 Min., die Stechborsten aus dem Blattgewebe zu ziehen. Jedoch verließen nur 12% in 25 Min die Pflanze, die übrigen liefen auf der Pflanze umher und siedelten wieder auf ihr.Das Alarmpheromon konnte in seiner Wirkung gefördert oder gehemmt werden durch zusätzliche, nichtchemische Reize. Bestimmte taktile Reize erhöhten die Wirkung des Siphonensekrets auf 100%. Andererseits verringerten Erschütterungen oder optische Reize die Reaktion. Larven vonCoccinella septempunctata L. beeinflußten die Reaktion der Läuse auf das Farnesen nicht, die Wirkungen durch Feinde und Farnesen erwiesen sich als additiv.(E)--Farnesen scheint nicht die einzige Komponente im Alarmpheromon der Erbsenlaus zu sein, denn der Anteil der Fallreaktionen an der Gesamtreaktion war beim (E)--Farnesen, unabhängig von seiner Konzentration, kleiner als bei Siphonensekret.

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Summary High levels of (E)--farnesene caused 84% of apterous pea aphids to withdraw their stylets from the plant tissue within 10 min. But only 12% left the plant within 25 min, the others were running on the plant and settled down again.The effect of alarm pheromone could be decreased and increased with additional, non-chemical stimuli. Special tactile stimuli increased the effect of siphunculi droplets secretion. On the other hand, vibration or optical stimuli decreased the reaction of the aphids. Larvae ofCoccinella septempunctata L. did not influence the reaction to farnesene, the escape responses to enemies and to farnesene seem to be additive.(E)--farnesene is probably not the only component in the alarm pheromone of the pea aphid as the percentage of reacting aphids showing the falling response was lower with (E)--farnesene —independent of the concentration — than with the siphunculi fluid.

     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

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.

     

Biosynthesis of gallotannins. Enzymatic conversion of 1,6-digalloylglucose to 1,2,6-trigalloylglucose

Cell-free extracts from Rhus typhina L. (staghorn sumach) leaves were found to catalyze the transfer of the galloyl moiety of -glucogallin (1-O-galloyl--D-glucose) to 1,6-di-O-galloyl--D-glucose, resulting in the specific formation of 1,2,6-tri-O-galloyl--D-glucose, an intermediate of gallotannin biosynthesis. The reaction product was unequivocally identified by co-chromatography with authentic references using reversed-phase high-performance liquid chromatography and by ¹H-nuclear-magnetic-resonance spectroscopy.Abbreviations HPLC high-performance liquid chromatography - NMR nuclear magnetic resonance     

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.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

NMR structure of the ribosomal protein L23 from Thermus thermophilus

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel -strands and three -helices connected as ------, and a large and flexible loop inserted between the third and fourth -strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.     

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.     

Synthesis of deoxygalactose-containing sialyl LeX ganglioside analogues to elucidate the structure necessary for selectin recognition

Sialyl Lewis X ganglioside analogues containing 4-deoxy-, 6-deoxy-, and 4,6-dideoxy-d-galactopyranose in place ofd-galactopyranose have been synthesized. Glycosylations of 2-(trimethylsilyl)ethyl 2,6-di-O-benzyl--d-galactopyranoside and 2-(trimethylsilyl)ethyl -d-fucopyranoside with the phenyl 2-thioglycoside derivative of sialic acid, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid (TfOH) as the promoter in acetonitrile, gave the desired 2-(trimethylsilyl)ethyl sialyl--(23)--d-galactopyranoside and--d-fucopyranoside, respectively. The sialylgalactose derivative obtained was then modified to 4-deoxy and 4,6-dideoxy derivatives. These were converted, byO-benzoylation, transformation of the 2-(trimethylsilyl)ethyl group to trichloroacetimidates, and introduction of the methylthio group with methylthiomethysilane, into the corresponding glycosyl donors, which were then coupled with 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(13)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(13)-2,4,6- tri-O-benzyl--d-galactopyranoside in the presence of dimethyl(methylthio)sulfonium triflate (DMTST). The resulting pentasaccharides were each converted to the corresponding -trichloroacetimidates, which, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the desired sphingosine derivatives. Selective reduction of the azide group,N-acylation with octadecanoic acid,O-deacylation, and saponification of the methyl ester afforded the target compounds.Synthetic Studies on Sialoglycoconjugates, Part 79.     

Zur Cytochemie der Lysosomen in der Rattenniere unter normalen und experimentellen Bedingungen

Zusammenfassung Es wird das Vorkommen der sauren Phosphatase (sP) und der -Glucu-ronidase (-Glu) in den Lysosomen von Rattennieren vom 16. Embryonaltag (ET) bis 33. Lebenstag (LT) sowie von erwachsenen unbehandelten, kastrierten und nach der Kastration mit gleich- bzw. gegengeschlechtlichen Hormonen behandelten Tieren untersucht. Bis zur Geburt entwickelt sich die lysosomale sP und -Glu in den einzelnen Nephronabschnitten und Sammelrohren annähernd parallel. Am 1. LT nimmt in den S₁-Segmenten der iuxtamedullären Nephrone Zahl, Durchmesser und Aktivität der sP positiven Lysosomen sprunghaft zu, um zwischen 5. und 9. LT wieder schnell abzunehmen. Die Aktivität der -Glu steigt dagegen kontinuierlich an. Zwischen 24. und 25. LT nimmt die Aktivität der -Glu in den Lysosomen der S₁- und S₃-Segmente ab, die der sP deutlich zu. Geschlechtsunterschiede treten bei der sP zum ersten Mal am 18. LT in den S₃-Segmenten, bei der -Glu am 25. LT in den S₁-Segmenten auf. Das ausdifferenzierte Enzymmuster für sP und -Glu kann erst bei erwachsenen Tieren nachgewiesen werden. Dann fallen bei beiden Geschlechtern unter den Lysosomen der S₁-Segmente zwei Größenklassen besonders auf: a. große Lysosomen (beim Weibchen 7 m, beim Männchen 5 m) und b. kleinere Lysosomen (bei beiden Geschlechtern 2–3 m). Die großen Lysosomen sind bei Weibchen zahlreicher und sP aktiver als bei Männchen. In den S₂-Segmenten (Durchmesser der Lysosomen 1,5–2,5 m) ist die -Glu in den Lysosomen der Männchen aktiver als bei den Weibchen; jedoch sind die Geschlechtsunterschiede in S₂ geringer als in S₁ In den S₃-Segmenten sind sP und -Glu in den Lysosomen weiblicher Nieren aktiver als in denen männlicher Tiere. — In den übrigen Abschnitten des Nephrons und in den Sammelrohren bestehen keine Geschlechtsunterschiede. — Die Lysosomen der Sammelrohre haben eine höhere Aktivität für -Glu als für sP.Durch Kastration werden die Geschlechtsunterschiede geringer, bleiben aber grundsätzlich erhalten; in den S₁-Segmenten können jedoch Lysosomen über 2,5 m nicht mehr nachgewiesen werden. Nach Testosteronbehandlung männlicher Kastrate nimmt die Aktivität der sP und -Glu gegenüber unbehandelten Kastraten ab. Bei kastrierten weiblichen Tieren treten nach Testosteronbehandlung wieder große Lysosomen auf, das Enzymmuster unbehandelter Tiere wird jedoch nicht erreicht. Östradiolbehandlung kastrierter Tiere führt bei beiden Geschlechtern in etwa zu einer Restitution der normalen Enzymverteilung, insbesondere bei den Männchen. Bei den Weibchen sind die Lysosomen größer und reagieren intensiver als bei Normaltieren, die Anzahl der Lysosomen scheint aber geringer zu sein.Insgesamt ergibt sich, daß die Lysosomen und die lysosomalen Enzyme unter dem Einfluß der Geschlechtshormone stehen.

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On the cytochemistry of lysosomes in the rat kidney under normal and experimental conditions

Summary Acid phosphatase (ap) and -glucuronidase (-glu) have been investigated in the lysosomes of the rat kidney between the 16th embryonic and 33th postnatal day. Furthermore these enzymes were demonstrated in the kidney of adult normal as well as of orchiectomized or ovarectomized rats following treatment with homosexual and heterosexual hormones. — Up till birth, the development of ap and -glu runs nearly parallel to one another in the nephron and in the collecting tubules. At the first day of life an irregular increase with respect to number, diameter, and activity of lysosomes containing ap can be observed in the S₁ segments of the iuxtamedullary nephrons; a decrease occurs between the 5th and 9th postnatal day. On the contrary the -glu activity increases continously. Between the 24th and 25th day of life its activity decreases in the lysosomes of the S₁ and S₂ segments; the ap activity, however, increases. For the first time sex-specific differences concerning the distribution pattern of ap can be revealed around the 18th day of life in the S₃ segments; in the case of -glu ca one week later in the S₁ segments. The final ap and -glu pattern only exists in the kidney of adult animals. Here, in the male and female kidney two classes of lysosomes exhibit striking peculiarities in the S₁ segments: a. big lysosomes (in male 5 m, in female 7 m in diameter) b. small lysosomes (in both sexes 2–3 m in diameter). The big lysosomes are more numerous. Moreover their ap activity is higher in female rats in comparison with the male kidney. In the S₂ segments (lysosomal diameter 1.5–2.5 m) the -glu activity of male rats surpasses that of females. However sex differences in the S₂ are as obvious as in the S₁ segments. In S₃ the strongest ap and -glu reaction appear in female kidneys. — In the other parts of the nephron and in the collecting tubules sexspecific differences have never been observed. — In the lysosomes of the collecting tubules more -glu than ap activity can be detected.Castration induces only a decrease of sex differences, but they do not disappear completely; in the S₁ segments lysosomes being bigger than 2.5 m are absent. In male castrates treated with testosterone the activity of ap and -glu is lowered in comparison with unsubstituted animals. Following application of testosterone the big lysosomes reappear in overectomized rats; but the enzyme pattern of untreated animals will never be obtained. In both sexes treatment of castrated animals with estradiol is accompanied by restitution of the normal ap and -glu pattern especially in males. In the female kidney lysosomes are bigger and more active in comparison with the controls; the number of lysosomes seems to be reduced. Summarizing strong evidence occurs that in the rat kidney lysosomes and lysosomal enzymes are controlled by sex hormones.

     

A guanosine 5′-triphosphate-dependent protein kinase is localized in the outer envelope membrane of pea chloroplasts

A guanosine 5-triphosphate (GTP)-dependent protein kinase was detected in preparations of outer chloroplast envelope membranes of pea (Pisum sativum L.) chloroplasts. The protein-kinase activity was capable of phosphorylating several envelope-membrane proteins. The major phosphorylated products were 23- and 32.5-kilo-dalton proteins of the outer envelope membrane. Several other envelope proteins were labeled to a lesser extent. Following acid hydrolysis of the labeled proteins, most of the label was detected as phosphoserine with only minor amounts detected as phosphothreonine. Several criteria were used to distinguish the GTP-dependent protein kinase from an ATP-dependent kinase also present in the outer envelope membrane. The ATP-dependent kinase phosphorylated a very different set of envelope-membrane proteins. Heparin inhibited the GTP-dependent kinase but had little effect upon the ATP-dependent enzyme. The GTP-dependent enzyme accepted phosvitin as an external protein substrate whereas the ATP-dependent enzyme did not. The outer membrane of the chloroplast envelope also contained a phosphotransferase capable of transferring labeled phosphate from [-³²P]GTP to ADP to yield (-³²P]ATP. Consequently, addition of ADP to a GTP-dependent protein-kinase assay resulted in a switch in the pattern of labeled products from that seen with GTP to that typically seen with ATP.Abbreviations GDP (GMP, GTP) guanosine 5-diphosphate (mono-, tri-); kDa-kilodalton - S_0.5 concentration of substrate supporting half-maximal velocity - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tricine N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine     

Mitochondrial and chloroplast targeting sequences in tandem modify protein import specificity in plant organelles

Protein targeting to plant mitochondria and chloroplasts is usually very specific and involves targeting sequences located at the amino terminus of the precursor. We challenged the system by using combinations of mitochondrial and chloroplast targeting sequences attached to reporter genes. The sequences coding for the presequence of the mitochondrial F₁-ATPase -subunit and the transit peptide of the chloroplast chlorophyll a/b-binding protein, both from Nicotiana plumbaginifolia, were fused together in both combinations, then linked to the reporter genes, chloramphenicol acetyl transferase (CAT) and -glucuronidase (GUS), and introduced into tobacco. Analysis of CAT and GUS activities and proteins in the subcellular fractions revealed that the chloroplast transit peptide alone was not sufficient to target the reporter proteins to chloroplasts. However, when the mitochondrial -presequence was inserted downstream of the chloroplast sequence, import of CAT and GUS into chloroplasts was observed. Using the reciprocal system, the mitochondrial presequence alone was able to direct transport of CAT and, to a lesser extent, GUS to mitochondria; the GUS targeting to mitochondria was increased when the chloroplast targeting sequence was linked downstream of the mitochondrial presequence. Immuno-detection experiments using subcellular fractions confirmed the results observed by enzymatic assays. These results indicate the importance of the amino-terminal position of the targeting sequence in determining protein import specificity and are considered within the hypothesis of a co-translational protein import.     

Enzymic synthesis of lacto-N-triose II and its positional analogues

N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc₂) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc₂, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC₆H₄NO₂-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC₆H₄NO₂-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose - NAHase N-acetylhexosaminidase - -CD -cyclodextrin     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

Improved in situ β-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of -galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) with 5-bromoindolyl--o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial -galactosidase (lacZ). After -galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for -galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of -galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.     

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.