Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

ATTRACTION D‘ACROLEPIOPSIS ASSECTELLA,EN OLFACTOMÈTRE,PAR DES SUBSTANCES ALLÉLOCHIMIQUES VOLATILES D’ALLIUM PORRUM

Résumé Pour rechercher le poireau, Acrolepiopsis assectella doit utiliser des stimulus olfactifs issus du végétal-hôte et notamment les substances soufrées spécifiques. Le pouvoir attractif de l'«odeur» du poireau et celui de différents thiosulfinates et disulfures trouvés dans cette plante sont donc étudiés et comparés par olfactométrie. Le comportement de mâles et de femelles vierges et fécondées est observé, en fonction de l'âge des imagos, pendant la scotophase. Alors qu'un courant d'air sans «odeur» significative déclenche essentiellement l'orientation et l'immobilisation des insectes face au flux, la plus part des mâles et des femelles quel que soit leur état sexuel sont attirés par le courant d'air lorsque celui-ci transporte l'«odeur» du poireau ou les substances volatiles étudiées. La sensibilité à l'«odeur» de la plante-hôte semble se modifier avec l'âge des insectes, les mâles âgés et les jeunes femelles étant les plus attirés. Cependant, ces dernières conservent une forte sensibilité une fois fécondées. Aux âges où l'«odeur» du poireau est la plus efficace, les thiosulfinates déclenchent chez les mâles et les femelles vierges une attraction plus importante que les disulfures, le radical propyle étant plus actif que le méthyle. Le thiosulfinate de dipropyle, labile, très abondant dans le poireau, semble donc avoir un rôle informationnel prépondérant.

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Summary To find the leek, Acrolepiopsis assectella uses olfactory stimulants from the hostplant, and specially the specific sulphur compounds. The attractiveness of the leek smell and of different thiosulfinates and disulfides found in this plant were studied and compared in an olfactometer. The behaviour of , and virgin or mated was observed during the scotophase according to to the age of the adults. In a clean air current the insects orient and remain stationary. The majority of and virgin or mated move upwind when the air current bears the leek smell or the voltile sulphur compounds. The sensitivity to the hostplant smell seems to change with the age of the insect, old and young being most attracted. Once mated, the retain a high sensitivity. When the responsiveness to the leek smell is highest (at 1 day and 5 days) the thiosulfinates have a greater effect than the disulfides in both and virgin . The propyl moiety is more active than the methyl one. The labile dipropyl thiosulfinate (propyl propanethiosulfinate), very profuse in the leek, seems to have the major role.

     

Hydrolysis of 2-substituted aryl and heteroaryl alkanoates by Candida rugosalipase

sThe hydrolysis of several esters catalyzed by lipase of Candida rugosa was used to analyse the interaction between the ester substrate and the active site and the geometry and steric restrictions of alk, ar and h subsites. Using 4-R-C H -CH(R)-COOEt and ethyl trans-cinnamate, the presence of a low electronic density zone in the ar pocket is described, the dimensions of the pocket produced by Phe-296 and Leu-297 in the alk subsite and the size of the h subsite are described.     

Ecological theories and Dutch nature conservation

This paper aims to achieve insight into various ecological theories in the Netherlands which have different, and sometimes opposing, views on the conservation of nature. Interviews, publications and archival research brought to light four separate theories: vitalistic/holistic, dynamic, cybernetic and chaos. Diversity is reached through stability according to vitalistic/holistic and cybernetic theories, but through change and instablility according to the dynamic and chaos theories. These two groups are working apart, and continue to have their own ideas. Prediction of the future is only possible with the vitalistic/holistic and cybernetic theories. Ecologists who adhere to these theories feel responsible and able in different ways to change ecological nature towards desirable end goals. The other two theories, dynamic and chaos, appear to be less activist.     

Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue

Summary An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a direct fluorescence procedure using FTC-toxin, but not by an indirect procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the direct and indirect procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the indirect procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a direct procedure using FTC-antiprofilaggrin but only weakly by an indirect double antibody procedure. Insensitivity to indirect procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.     

Structure granaire,réduction du NADP et photophosphorylation des chloroplastes isolés de feuilles d'orge

Summary Barley chloroplasts (Hordeum vulgare L.) have been isolated in aqueous medium by previously described techniques which preserve the outer plastid membrane in numerous chloroplasts. Thus intact chloroplasts are concentrated in Class I fraction and non intact in Class II fraction. A third type, broken Class I, is obtained by the action of a very dilute buffer solution.The observation of freeze-etched preparations has shown that the main distinctive character between the two classes is not so much the presence or the absence of the outer plastid membrane, as it was assumed, as the state of the thylakoids. In Class I chloroplasts grana are shrunken, in those of Class II, grana are swollen. In both cases the outer membrane may or may not be preserved.The breakage of chloroplasts resulting from treatment with osmotic shocks is due to the dilatation of the extra-lamellar matrix. The swelling of intra-thylakoidal volumes resulting from such a treatment is much less important.It is shown that Class I chloroplasts have a very low NADP reductase activity which is strongly increased after the chloroplasts are broken by osmotic shocks. Such a treatment is without any effect on Class II chloroplasts, which always exhibit a low NADP reductase activity.The situation is rather different in regard to photophosphorylation. It is also lower in Class II than in Class I, but after breakage by osmotic shocks the increase of this activity is weak, and much less significant than the increase observed in NADP reduction. The compression and cohesion of the thylakoids is not required for high photophosphorylation activity with artificial electron acceptors such as PMS. In this case cyclic photophosphorylation is higher in Class II than in Class I.

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Abbreviations ADP, ATP adénosine di et triphosphate - EDTA éthylène diamine trétaacétate - NAD, NADP, NADPH₂ nicotinamide adénine dinucléotide, d° phosphate (formes oxydée et réduite) - PMS phénazine méthosulfate - Pi phosphate inorganique     

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms viability and culturability are often equated. However, in recent years the apparently self-contradictory expression viable-but-nonculturable (VBNC) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms viability and culturability in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces VBNC with expressions that are internally consistent.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Selection for rate of development and pupal weight in ‘Cnephasia’ jactatana: A genetic tool for quality improvement

Selection for fast and slow rate of pupation over three successive generations in Cnephasia jactatana (Walker) (Lepidoptera: Tortricidae) produced a fast strain that pupated 2 days earlier than the slow strain. Selection for pupal weight was asymmetrical with the light selection having more effect than the heavy selection. The larval critical weight (L_CW), the larval maximum weight (L_MW) and the latent feeding period were not affected by any of the selections. Slow and light selections resulted in longer pre-pupal periods than fast, heavy or the laboratory population. Fecundity was reduced in females from slow and light selections. Selection in insects is discussed as a possible genetic tool for overall quality improvement.Résumé La sélection pour la rapidité d'apparition des chrysalides de C. jactatana Walker produit en 3 générations une lignée rapide qui se nymphose 2 jours avant la lente. La sélection pour le poids des chrysalides est asymétrique, ayant plus d'effet sur la sélection développement lent que sur la sélection développement rapide. Le poids critique larvaire (L_CW), le poids larvaire maximal (L_MW) et la période latente d'alimentation ne sont pas modifiés par ces sélections. Les lignées lente et légère ont des périodes prénymphales plus longues que les lignées rapide et lourde, ou la souche de laboratoire. Les fécondités des lignées lente et légère sont réduites. Les sélections associées des insectes sont examinées comme outil génétique potentiel permettant d'améliorer les performances globales.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2-N-acetylglucosaminyl-transferase I using synthetic substrate analogues

UDP-GlcNAc: Man3R 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M _r 42,000). TheV _max for the pure enzyme with [Man6(Man3)Man6](Man3)Man4GlcNAc4GlcNAc-Asn as substrate was 4.6 µmol min^–1 mg^–1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in 1–2 linkage to the Man3Man-terminus of the substrate. Several derivatives of Man6(Man3)Man-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the -linked mannose of Man6(Man3)Man-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the 3-linked mannose (Man) of the substrate increases theK _M 20-fold. Modifications on the 6-linked mannose or on the core structure affect mainly theK _M and to a lesser degree theV _max, e.g., substitutions of the Man6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK _M, whereas various other substitutions at the 3-position increase theK _M slightly. Man6(Man3)4-O-methyl-Man4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.Abbreviations BSA Bovine serum albumin - Bn benzyl - Fuc, F l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - Glc d-glucose - GlcNAc, Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man, M d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈ COOOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - NMR nuclear magnetic resonance - PMSF phenylmethylsulfonylfluoride - pnp p-nitrophenyl - SDS sodium dodecyl sulfate - T transferase - Tal d-talose - Xyl d-xylose; - {0, 2 + F} Man6 (GlcNAc2Man3) Man4GlcNAc4 (Fuc6) GlcNAc - {2, 2} GlcNAc2Man6 (GlcNAc2Man3) Man4GlcNAc4GlcNAc; M₅-glycopeptide, Man6 (Man3) Man6 (Man3) Man4 GlcNAc4GlcNAc-Asn Enzymes: GlcNAc-transferase I, EC 2.4.1.101; GlcNAc-transferase II, EC 2.4.1.143; GlcNAc-transferase III, EC 2.4.1.144; GlcNAc-transferase IV, EC 2.4.1.145; GlcNAc-transferase V, UDP-GlcNAc: GlcNAc2 Man6-R (GlcNAc to Man) 6-GlcNAc-transferase; GlcNAc-transferase VI, UDP-GlcNAc: GlcNAc6(GlcNAc2) Man6-R (GlcNAc to Man) 4-GlcNAc-transferase; Core 1 3-Gal-transferase, EC 2.4.1.122; 4-Gal-transferase, EC 2.4.1.38; 3-Gal-transferase, UDP-Gal: GlcNAc-R 3-Gal-transferase; blood group i 3-GlcNAc-transferase, EC 2.4.1.149; blood group I 6-GlcNAc-transferase, UDP-GlcNAc: GlcNAc3Gal-R (GlcNAc to Gal) 6-GlcNAc-transferase.     

Localization of a gene for human α-galactosidase B (=N-Acetyl-α-D-Galactosaminidase) on chromosome 22

Summary The localization of the structural gene for human -galactosidase B (=N-acetyl--galactosaminidase) was investigated by means of man-Chinese hamster and man-mouse somatic cell hybrids. The hybrid clones were analyzed for chromosomes and for a large number of known enzyme markers. The lysates of the hybrid cells were treated with Sepharose-coupled antihuman -galactosidase B and the activity of the adsorbed enzyme was measured on the Sepharose beads as N-acetyl--galactosominidase. The results show that the structural gene for human -galactosidase B is situated on chromosome 22, and that there is no structural relationship between human -galactosidase A and human -galactosidase B.     

Ontogeny of estrogen receptor (ER) alpha and its co-localization with pituitary hormones in the pituitary gland of chick embryos

Estrogen is involved in regulating the development and hormone secretion of the anterior pituitary gland following its binding to estrogen receptors (ERs) expressed on pituitary cells. However, the pituitary is comprised of several cell types, and to date, there is no data about the specific cell types expressing ERs in embyonic chick pituitary. We therefore followed, by immunohistochemistry, the ontogeny of the pituitary ER alpha (ER), and the cell types expressing ER throughout chick embryo development. ER immunoreacitivity was restricted to the nuclei of pituitary cells. ER-immunopositive (ER⁺) cells were first detected at embryonic day 6.5 (E6.5), after which ER⁺ cells were consistently detected throughout the anterior pituitary gland, although the density of ER⁺ cells in the caudal lobe of the pars distalis was higher than that in the cephalic lobe. The proportion of ER⁺ cells in the pituitary was about 6% at E8.5; expression increased to 22% by E18.5 of gestation, with no additional change until hatching. Double-labeling of ER and pituitary hormones showed that the dominant cell types expressing ER were gonadotrophs immunopositive for luteinizing hormone (LH); the proportion of ER⁺ cells expressing LH increased throughout gestation and reached approximately 57% at hatching. About 2%–6% of thyroid-stimulating-hormone-immunopositive and 1%–2% prolactin-immunopositive cells expressed ER at later stages of embryonic development, but no growth-hormone-positive or adrenocorticotropic-hormone-positive cells expressed ER during the embryonic period. Thus, gonadotrophs are the main cell population expressing ER in the anterior pituitary gland of chick embryo, and ER is involved in regulating the development of the pituitary gland and the maturation of the hormone-secreting function.This work was supported by grants from the Natural Science Foundation for Outstanding Young Scientists of China (30325034) and the Natural Science Foundation of China (30170693, 30471264).     

Temporal oviposition patterns of Heliothis zea and Spodoptera ornithogalli

Egg production under laboratory conditions was examined over the lifespan of Heliothis zea (Boddie) and Spodoptera ornithogalli Guenée (Lepidoptera: Noctuidae). Although H. zea oviposits singly and S. ornithogalli oviposits in masses, temporal trends were similar. Egg numbers peaked shortly after mating and then rapidly declined. Egg weights also peaked shortly after mating, but decreased gradually over time. Temporal oviposition patterns were more erratic for unmated than mated females, suggesting the importance of mating in establishing the shape of the oviposition curve.

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Résumé La production d'ufs d'Heliothis zea (Boddie) et de Spodoptera ornithogalli Guenée (Lepidoptera: Noctuidae) durant toute la vie adulte a été examinée au laboratoire. Bien que H. zea dépose ses ufs isolément et que S. ornithogalli les dépose en groupes, les courbes temporelles d'oviposition chez des individus fécondés sont similaires. Le nomber d'ufs culmine peu après l'accouplement, puis décline rapidement. Le poids des ufs culmine aussi peu après l'accouplement, puis décroît graduellement. Des femelles fécondées produisent environ deux fois autant d'ufs que les femelles non fécondées, quoique la longévité ne diffère pas significativement entre les deux groupes. Des femelles non fécondées montrent des distributions temporelles plus irrégulières que des femelles fécondées, en ce qui concerne le nombre des ufs et leur poids. Ces irrégularités ont été attribuées à la tendance des femelles non fécondées à retenir leur ufs, ce qui suggère que l'accouplement exerce une influence sur la forme de la courbe temporelle d'oviposition. Des femelles d'H. zea contenant un, deux ou trois spermatophores n'ont pas produit des nombres d'ufs significativement différents.

     

Hemoglobin Stanleyville II (α78 Asn→Lys) is associated with a 3.7-kb α-globin gene deletion

Summary A family with one homozygote and three heterozygotes for hemoglobin Stanleyville II (78 AsnLys) has been analyzed by -globin gene mapping. The pattern of restriction fragments with the enzymes BamHI and BglII demonstrated that the globin variant is associated with a 3.7-kb -globin gene deletion.     

Comparison of the homologous carboxy-terminal domain and tail of α-crystallin and small heat shock protein

The C-terminal domain and tail, which is the most conserved region of the -crystallin/small heat shock protein (HSP) family, was obtained from rat A-crystallin, bovine B-crystallin and mouse HSP25. All three domains have primarily -sheet conformation and less than 10% of -helix, like the proteins from which they are derived. Whereas the C-terminal part of A-crystallin forms dimers or tetramers, the corresponding regions of B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the -crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.Abbreviations A2Dt residues 64–173 of rat -crystallin - B2Dt residues 70–175 of bovine B-crystallin - bp base pair - HSP2Dt residues 92–209 of HSP25 - HSP(s) heat shock protein(s) - HSP25 mouse small HSP - PCR polymerase chain reaction - PMSF phenylmethylsulfonyl chloride - SDS sodium dodecyl sulfate; polyacrylamide - WSF water-soluble fraction     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.