Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Pumpkin (Cucurbita sp.) seed globulin III. Comparison of subunit structures among seed globulins of various Cucurbita species and characterization of peptide components

Various Cucurbita seed globulins showed patterns similar toone another on SDS-gel electrophoresis, and ß bandsfor unreduced globulins and , ', and ' bands for reduced ones.On gel electrophoresis in 6 M urea, reduced globulin gave twoacidic and two basic bands. These corresponded to and ' chainsand ₁ and ₂ chains, respectively, identified by two-dimensionalurea-SDS gel electrophoresis. The compositions of the and ßsubunits were proposed. (Received September 8, 1977; )     

Enzymatic Degradation of Chlorophyll in Chenopodium album

The breakdown of chlorophyll (Chi) in crude extracts of Chenopodiumalbum (white goose foot) in the dark was examined. Derivativesof pheophorbide were formed when Chi or chlorophyllide wasincubated with depigmented crude extracts. The formation ofpheophorbide was completely prevented by heat treatment of extracts,indicating that the reaction was enzymatic, and the presenceof a Mg-releasing enzyme, the so called Mg-dechelatase, waspostulated. This hypothesis was strongly supported by the observationthat the formation of pheophorbide was inhibited by 51% by 10mM MgCl₂. Analysis by high-performance thin-layer chromatography(HPTLC) and liquid chromatography (HPLC) showed that the appearanceof chlorophyllide , pheophorbide 13²-hydroxychlorophyllide and pyropheophorbide was accompanied by a concomitant decreasein levels of Chi The formation of 13²-hydroxychloro-phyllide was not clearly an enzymatic reaction and requires furtherexamination. It appears that Chl is degraded in a crude extractof C. album via the following enzymatically catalyzed reactions (Received September 10, 1990; Accepted November 15, 1990)     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

Induction of sexual agglutinability of {alpha} mating-type cells as the primary action of the peptidyl sex factor from {alpha} mating-type cells in Saccharomyces cerevisiae

The effect of a pure preparation of substance-I_A (S-I_A) whoseamino acid sequence is identical to that of one of the factorpeptides (2), on sexual agglutinability and DNA synthesis wascomparatively studied. The optimum concentration of S-I_A forthe induction of sexual agglutinability of cells of an inducible strain was 1 ng/ml. The inducing action of S-I_A was detectedin 20 min and reached a maximum in 60 min. Only 8.7% inhibitionof DNA synthesis by S-I_A in the same strain was detected in1 hr and 40.4% inhibition in 2 hr at a concentration of 1 µg/ml.These results suggest that the primary action of the peptidyl sex fractor on a mating-type cells is the induction of sexualagglutinabiity. (Received October 25, 1977; )     

An {alpha}-Glucan from Suspension-Cultured Rice Cells

An -glucan was isolated from 11-day-old suspension-culturedrice cells by extraction with hot Na-phosphate buffer (pH 6.8).The -glucan had []_D=+234? (C = 0.14, in water) and its averagemolecular weight was estimated to be about 1.4 ? 10⁴, basedon elution characteristics on acalibrated Sepharose CL-6B column.Upon partial acid hydrolysis, the -glucan gave mainly malto-oligosaccharides.The maximum absorption of the iodine complex of the -glucanin the presence of Na₂SO₄ was at 470 nm. The results of hydrolysisby , ß- and iso-amylases and methylation analysisindicated that the isolated -glucan is a highly branched polysaccharidewith an average chain length of 9. The exterior and interiorchain lengths of the -glucan were calculated to be 5 and 3,respectively. (Received July 23, 1986; Accepted February 7, 1987)     

Pumpkin (Cucurbita sp.) seed globulin IV. Terminal sequences of the acidic and basic peptide chains and identification of a pyroglutamyl peptide chain

Pumpkin seed globulin is composed of heterogeneous polypeptidechains, acidic and chains and basic ₁ and ₂ chains (12). This study showed that the basicchains had similar N-terminal sequences, Gly-Leu-Asp-Glu-Thr-Ile-for the ₁ chain and Gly-Leu-Glu-Glu-Thr-Ile- for ₂. On the contrary,the N-terminal sequences of the acidic and chains were dissimilar, Ile-Gln-Gly-Tyr- for the chain and no N-terminal residue for the chain, according to routine terminal analysis. Pyrrolidonylpeptidase digestion of the chain and its thermolysin digestion followed by Edman degradationsrevealed that the N-terminal sequence of the chain was < Glu-Ile-Glu-Gln-Gln-Glu-Pro(Trp,Ser)-. The N-terminal sequences and the C-terminal residuesindicated that the acidic and chains were more heterogeneous than the basic ₁ and ₂ chains.A preliminary study on the degradation of storage globulin isalso presented. (Received November 9, 1979; )     

Regulation of sexual agglutinability in Saccharomyces cerevisiae of {alpha} and {alpha} types by sex-specific factors produced by their respective opposite mating types

The sexual agglutinability of haploid cells of heterothallicSaccharomyces cerevisiae was repressed when they were culturedin the absence of easily fermentable sugars, such as glucoseand mannose. The repression was reversed by the action of hormone-likesubstances of the opposite mating types. The substance producedby mating type cells was identical to subtsance-I which isknown to induce sexual agglutinability of inducible matingtype cells. The mating type cells produce a new hormone-likesubstance which induces or enhances sexual agglutinability of mating type cells. A crude fraction of the mating type-specific substance ( substance-I)was obtained by passing the culture filtrate of mating typecells through Amberlite CG-50 (H⁺ form), followed by elutionwith 1.5 M ammonia. ² On leave from Osaka City University. (Received December 25, 1975; )     

Promotion and inhibition of {alpha}-amylase production in barley endosperm by cyclic 3',5'-adenosine monophosphate and adenosine diphosphate

The possibility that gibberellin-induced -amylase synthesisin barley endosperm might be mediated by cyclic-3',5'-adenosinemonophosphate (3',5'-AMP) was examined. Promotion of -amylasesynthesis by 3',5'-AMP (5 mM) was observed in the absence ofgibberellic acid (GA₃) and in combination with GA₃ at concentrationsbelow 2 mµM. When combined with gibberellin at concentrationsabove 2 mµM, however, 3',5'-AMP reduced the amount of-amylase obtained. The cyclic nucleotide showed slight activityat concentrations as low as 0.05 mM. These promotions were shownto be due to increased synthesis of -amylase rather than toan increased secretion of the enzyme. Of a variety of adeninecompounds and nucleoside diphosphates tested only 3',5'-AMPand adenosine diphosphate (ADP) induced -amylase synthesis.Longer incubation times were necessary to obtain maximal -amylaseinduction with the nucleotides than with GA₃. ADP and 3',5'-AMPwere about one third and one fifth as active, respectively,as GA₃ in promoting -amylase synthesis, although GA₃ was morethan 10⁷ times more effective. AMO-1618 did not inhibit theaction of the nucleotides and methanolic extracts of the nucleotidesshowed no gibberellin-like activity. Both nucleotides were synergisticwith GA₃ in overcoming the inhibitory effects of acetate andcitrate buffers on -amylase synthesis. (Received February 24, 1969; )     

An NTP-Binding Protein That Cross with a Ras-Specific Antibody in the Myceia of Neurospora crassa

A Ras-related NTP-binding protein was partially purified froma membrane fraction derived from the mycelia of Neurospora crassa.[-³²P]ATP and [-³²P]GTP were incubated with mem brane and solublefractions which were then irradiated with UV light to inducecrosslinking of tightly bound nucleotides. After SDS-polyacrylamidegel electrophoresis, blotting onto a nitrocellulose filter andautoradiography it was apparent that most of the proteins thatbound [-³²P]-GTP also bound [-³²P]ATP. Pretreatment of the membranefraction with Ras-specific antibody effectively blocked thebinding of [-³²P]ATP and [-³²P]GTP to several ATP-GTP-bindingproteins. The band of a protein with a molecular weight of 26kDa on the SDS-polyacrylamide gel cross-reacted strongly withthe Ras-specific antibody. The protein was extracted from thegel and further purified by repeated gel electrophoresis. Thepurified protein bound [-³²P]ATP, [-³²P]-GTP, [-³²P]CTP and[-³²P]UTP at 1.6x10^– M and was autophosphorylated in thepresence of [-³²P]ATP and [-³²P]GTP at 1.7x10 M. Pretreatmentof the protein with Ras-specific antibody partially blockedthe autophosphorylation in the presence of these nucleotides.The binding of [-³²P]ATP to the NTP-binding protein was blockedby addition of ATP at 10^–4–10^–3 M. ATP ata concentration of 10^–4 M prevented the binding of [-³²P]to a greater extent than did GTP at the same concentration.Binding of [-³²P]CTP and [-³²P]UTP to the protein was also observed. (Received October 7, 1991; Accepted July 14, 1992)     

BACTERIAL BREAKDOWN OF {varepsilon}-CAPROLACTAM AND ITS CYCLIC OLIGOMERS

Eleven different types of bacteria were isolated which werecapable of growing on -caprolactam, the monomeric material fornylon 6 polyamide, as the sole source of both carbon and nitrogen. The optimal concentration of -caprolactam for the bacterialgrowth was about 0.6% in a synthetic liquid medium enrichedwith a small amount of yeast extract. The bacterial strains grew also on -butyrolactam, -valerolactamand the -amino acids corresponding to these lactams and -caprolactam.Ammonium adipate was a good substrate for the growth of allthe strains. One strain of Corynebacterium aurantiacum was found to be capableof utilizing cyclic and linear oligomers of 6-aminocaproic acidwith an exception of cyclic dimer. The strains of corynebacteria required vitamin B₁ for growth. Metabolism of -caprolactam and related compounds is discussedbriefly. (Received September 9, 1965; )     

Purification by Affinity Chromatography of {alpha}-Amylase--A Main Amylase in Cotyledons of Germinating Vigna mungo Seeds

In Vigna mungo cotyledons, the -amylase activity increased markedlyduring germination at 27°C in the dark, while the activityof other amylases was very low. The -amylase was purified from4-day-old cotyledons by affinity chromatography on epoxyactivatedSepharose 6B substituted with rß-cyclodextrin andby column chromatography on Bio-Gel P-200. Gel filtration andpolyacrylamide gel electrophoresis showed that the enzyme existsmostly as a monomer (43,000 daltons), but partially aggregatesto form dimer, trimer and further multimers. Ca²⁺ protectedthe -amylase against heat inactivation. Incubation of the enzymewith 5 mM EDTA or dialysis against 10 mM EDTA resulted in a50–90% loss of activity. The inactivation was partiallyreversed by the addition of Ca²⁺. Other properties, such asthe amino acid composition, K_m value, pH optimum and activationenergy were similar to those of other plant -amylases. (Received May 6, 1981; Accepted June 22, 1981)     

Effects of Metal Chelators on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after being detachedand kept in the dark was studied in terms of the loss of chlorophyll.Chlorophyll was more rapidly degraded in maize than hydrangeaduring the incubation period in the dark. The loss of chlorophyllin the dark was effectively inhibited in both plants by ,'-dipyridyland o-phenanthroline at concentrations between 0.01 and 0.1mM. Three other chelators of iron produced lesser inhibitionand only at higher concentrations. EDTA prevented the loss ofchlorophyll in maize leaves at concentrations above 10 mM, butdid not do so in hydrangea leaves. Detached leaves floated on EDTA, ,'-dipyridyl or o-phenanthrolinesolutions and exposed to light exhibited a marked bleaching.The bleaching was partially inhibited by applying ascorbic acid. (Received December 26, 1981; Accepted October 18, 1982)     

Sialylation of n-alkyllactosides, galactosides and glucosides by sialyltransferases from rat liver Golgi vesicles

nAlkyl - and -lactosides, galactosides and glucosides with differentalkyl chain lengths (C₂, C₈, C₁₄, and C₂₀) were synthesizedand used as acceptors for sialyltransferases from rat liverGolgi vesicles. The -galactosides, -glucosides, and both - and-lactosides, were sialylated. Keeping the acceptor concentrationconstant, sialylation rates reached a maximum for the n-octyl- and -lactosides, n-Octyl -galactoside and noctyl -glucoside,respectively. noctyl -glucoside, respectivwly. n-Octyl -galactosideand n-octyl -glucoside were not sialylated. The reaction productswere characterized by TLC. With n-octyl lactoside and galactosideas acceptors, two major sialylation products were formed. Thjeycould be separated by preparative TLC, and their structureswere identified as 2–3 and 2–6 sialylated acceptors,respectively, by a combination of periodated oxidation, NaBD₄reduction,permethylation and subsequent analysis by fast atombombardment mass spectrometry (FAB-MS). The structure of thesingle product obtained from n-ictyl -glucoside was determinedto be the 2–6 sialylated glucoside. Competition experimentswith n-octyl lactoside and lactosylceramide and gangliosideGal1-3GalNAc1-4(NeuAc2–3)Gal1–4Glcbeeta1–1Cer(GM1) as acceptors for sialyltransferases suggested that SAT-I[NeuAc2–3Gal1–4Glc1-1Cer (GM3) synthase] was atleast in least in part responsible for the 2–3 sialylationof n-octyl lactoside. alkylgalactosides alkylglucosides alkyllactosides neoglycolipids sialytransferases     

Hydrophobic mannosides act as acceptors for trypanosome {alpha}-mannosyltransferases

A series of hydrophobic mannosides were synthesized and testedfor their ability to act as acceptor substrates for mannosyltransferasesin a Trypanosoma brucei cell-free system. The thiooctyl -mannosidesand octyl -mannosides all accepted single mannose residues in-linkage, as judged by thin layer chromatography of the productsbefore and after jack bean -mannosidase digestion. The mannosylationreactions were inhibited by amphomycin, suggesting that theimmediate donor was dolicholphosphate-mannose (Dol-P-Man) inall cases. The transferred -mannose residues were shown to beboth 1-2 and 1-6 linked by Aspergillus phoenicis -mannosidaseand acetolysis treatments, respectively. These data suggestthat the compounds can act as acceptor substrates for the Dol-P-Mandependent 1-2 and 1-6 mannosyltransferases of the GPI biosyntheticpathway and/or the dolichol-cycle of protein N-glycosylation.One of the compounds, Man1-6Man1-O-(CH₂)₇CH₃, inhibited endogenousGPI biosynthesis in the cell-free system, suggesting that itcould be a substrate for the trypanosome Dol-P-Man:Man₂GlcN-Pl1-2 mannosyltransferase. dolichol glycosylphosphatidylinositol mannosyltransferase trypanosome     

A Transient Stimulation of Net Photosynthesis of Rye Leaves by {alpha}-Hydroxy-2-pyridinemethanesulphonic Acid ({alpha}-HPMS) due to Inhibition of Photorespiratory CO2 Release

The effects of -hydroxy-2-pyridinemethanesulphonic acid (-HPMS)upon net photosynthesis (P_n, the CO₂ compensation point (),post-lower illumination burst of CO₂ (PLIB) and post-lower temperatureburst of CO₂ (PLTB) in detached rye (Secale cereale L.) leaveswere investigated. At low concentrations ( 0.5 mol m^–3),-HPMS initially stimulated P_n and decreased the magnitude ofboth PLIB and PLTB. The decreased at all concentrations of-HPMS (0.05–5.0 mol m^–3. The effects of -HPMS onP_n and were time-dependent and, after a few minutes, the P_nwas inhibited while values increased considerably. At a higherconcentration (5.0 mol m ^–3), the transient effects of-HPMS were shorter () or not observed at all (P_n. Both PLIBand PLTB, when expressed in relation to P_n, increased at higherlevels of this compound. Similar data with respect to the effectsof -HPMS on PLIB and PLTB were found for leaves of dandelion(Taraxacum officinale L.). The results suggest that -HPMS may stimulate P_n by inhibitingphotorespiration, as originally suggested by Zelitch (1966),but only at low concentrations and over a short time span. Thedecrease of PLIB and PLTB values at low -HPMS levels is consistentwith these processes being a residual activity of the glycolatepathway. Key words: CO₂ compensation point, -hydroxy-2-pyridinemethanesulphonic acid, photorespiration, photosynthesis     

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle {psi} for the orientation of {alpha}1,6-arm

The conformation of the heptasaccharide Man-1,6-(Man-1,3)(Xyl-ß1,2)-Man-ß,4-GlcNAc₂-ß1,4-(L-Fuc-1,3)-GlcNAc₁,the carbohydrate moiety of Erythrina corallodendron lectin (EcorL),the hexasaccharide Man-1,6-(Man-1,3) (GlcNAc-ß1,4)-Man-ß1,4-GlcNAc-ß1,4-GlcNAcand their disaccharide fragments have been studied by moleculardynamics (MD) simulations for 1000 ps with different initialconformations. In the isolated heptasaccharide, the most frequentlyaccessed conformation during MD has a value of 180° aroundMan-1,6-Man linkage. This conformation is stabilized by theformation of a hydrogen bond between the carbonyl oxygen ofGlcNAc₂ with the O3/O4 hydroxyls of the 1,6-linked mannose residue.The conformation of the heptasaccharide found in the crystalstructure of the EcorL-lactose complex (Shaanan et al., Science,254, 862, 1991), that has a value of 76° around Man-1,6-Manlinkage, is accessed, although less frequently, during MD ofthe isolated oligosaccharide. The ,, = 58°,–134°,–60°conformation around Man-₁,6-Man fragment observed in the crystalstructure of the Lathyrus ochnrs lectin complexed with a biantennaryoctasaccharide (Table I in Homans,S.W., Glycobiology, 3, 551,1993) has also been accessed in the present MD simulations.These values for the 1,6-linkage, which are observed in theprotein-carbohydrate crystal structures and are accessed inthe MD simulations, though occasionally, have not been predictedfrom NMR studies. Furthermore, these different values of leadto significantly different orientations of the 1,6-arm for thesame value of . This contrasts with the earlier predictionsthat only different values of can bring about significant changesin the orientation of the ₁,6-arm. The MD simulations also showthat the effects of bisecting GlcNAc or ß1,2-xyloseare very similar on the ₁,3-arm and slightly different on the₁,6-arm. bisecting GlcNAc carbohydrates glycoprotein lectinsaccharide complex     

Premature Drying Increases the GA-Responsiveness of Developing Aleurone Layers of Barley (Hordeum vulgare L.) Grain

The effect of premature drying on the sensitivity of aleuronelayer cells of developing barley (Hordeum vulgare L.) grainto gibberellic acid (GA₃) was investigated. The capacity ofbarley aleurone layer cells to respond to GA₃, as indicatedby -amylase synthesis and secretion by de-embryonated grain,increased during the later stages of development. Aleurone layersof immature grain (younger than 30 d after anthesis; DAA) werenot capable of producing amylase in response to GA₃; however,premature drying at this stage promoted GA-responsiveness resultingin the induction of mRNA for -amylase and increased -amylasesynthesis and secretion. Preincubation of the immature grainor its maintenance at 100% relative humidity prior to exposureof the de-embryonated grain to GA₃ also led to an enhanced capacityof the aleurone layer to produce amylase and its mRNA as comparedto the fresh, untreated grain. However, the amount of mRNA andenzyme produced was much lower than that induced by prematuredrying. Moreover, following these nondrying treatments, thealeurone layer cells remained unresponsive to exogenous GA₃;the same amount of enzyme was produced in the absence of appliedGA₃. Transient expression of chimeric gene constructs in aleuronelayer cells of de-embryonated grain suggest that drying up-regulatesthe -amylase gene promoter in response to GA₃. We conclude thatdesiccation is required for barley aleurone layer cells to becomeresponsive to GA₃ and hence express their full potential foramylase synthesis and secretion. ³Present address: Department of Biochemistry, University ofMissouri, 117 Schweitzer Hall, Columbia MO 65211, U.S.A.     

A Mutation Leading to Constitutive Expression of High Sexual Activities in the Yeast Saccharomyces cerevisiae

An a mating type mutant of the yeast Saccharomyces cerevisiaewhich expressed high sexual activities during vegetative growthwas isolated and characterized. Its constitutive sexual agglutinabilitywas higher than the sexual agglutinability of its parental straininduced by pheromone. It produced a pheromone and -pheromone-inactivatingsubstances in larger amounts than its parental strain. It alsoproduced large pear-shaped cells (shmooed cells) without pheromone,was more sensitive to pheromone, and grew vegetatively moreslowly than its parental strain. When the mutant was crossedto a wild type strain isogenic with the parental strain, amating type segregants with high constitutive sexual agglutinabilityshowed self-shmooing. However, in a mating type segregants self-shmooingwas not observed regardless of the degree of their sexual agglutinability.The cross between a and segregants, both of which carried themutation, had higher frequency of zygote formation than thecrosses between a and cells one of which or both of which wereof wild type. (Received September 9, 1985; Accepted February 8, 1986)     

ON THE EFFECTS OF PLANT EXTRACTS ON THE {alpha}-AMYLASE ACTIVITY IN RICE ENDOSPERM

Effects of ethanol extracts obtained from several plant materialson -amylase activity in rice endosperm were examined. Promotingeffect on the -amylase activity was estimated in terms of gibberellinA₃ equivalence using the embryoless endosperm. Inhibiting effectwas examined using intact seed. On chromatography of the extractfrom Prunus, the zones showing promotion of the -amylase activityalso exhibited the activity for the elongation of the leaf sheathof rice seedling. But this was not the case with the extractsfrom Pharbitis, Allium and Lupinus. (Received April 13, 1966; )