Identification and differential accumulation of two isoforms of the CF1-beta subunit under high light stress in Brassica rapa.

The chloroplast ATP synthase coupling factor CF1 complex contains five nonidentical subunits, alpha, beta, gamma, delta, and epsilon, with a stoichiometry of 3:3:1:1:1. The beta subunit contains the catalytic site for ATP synthesis during photooxidative phosphorylation in the chloroplast. In this study, we have identified two isoforms of the CF1-beta subunit at 56 and 54 kDa in the chloroplast of Brassica rapa, through isolation/purification, proteolytic digestion and internal peptide sequencing. Examining their accumulation pattern demonstrates that both isoforms coexist during chloroplast biogenesis and in mature thylakoid membranes, but the 54 kDa isoform is more apparently upregulated by light or under light stress. LiDS-PAGE shows that the 56 kDa is a major isoform of the CF1-beta subunit under normal light conditions, and its amount was not influenced during high light or other light stress treatments. The 54 kDa isoform is a minor band at normal conditions; however, it significantly increased under excess light stresses, such as high or low light with drought and/or high temperature. Particularly, a ninefold increase was observed after 8-10 h of high light treatment with drought and high temperature. The results suggest that light stress induction of the 54 kDa CF1-beta isoform may present a positive response during chloroplast photoacclimation.     

Purification and characterization of mycoferritin from Fusarium verticillioides MRC 826

The fungus Fusarium verticillioides MRC 826 (ascomycetes species), a toxigenic isolate is capable of synthesizing mycoferritin only upon induction with iron in yeast extract sucrose medium. The molecular mass, yield, iron and carbohydrate contents of the purified mycoferritin were 460 kDa, 0.010 mg/g of wet mycelia, 1.0 and 40.2%, respectively. Native gel electrophoresis of the mycoferritin revealed two bands possibly representing isoforms of ferritin. Subunit analysis by SDS–PAGE showed a single protein subunit of ~24 kDa suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy revealed an apparent particle size of 100 Å. N-terminal amino acid sequencing of mycoferritin showed identities with other eukaryotic ferritin sequences. The spectral characteristics were similar to equine spleen ferritin. However, circular dichroic spectra revealed a higher degree of helicity. Functionally, induction of mycoferritin minimizes the pro-oxidant role of iron.     

Purification and specificity of two alpha-glucosidase isoforms of the parasitic protist Trichomonas vaginalis

Two isoforms of alpha-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.     

Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle

This study focuses on the effects ofmechanical unloading of rat soleus muscle on the isoform patterns ofthe three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI),and troponin C (TnC). Mechanical unloading was achieved by hindlimbunloading (HU) for time periods of 7, 15, and 28 days. Relativeconcentrations of slow and fast TnT, TnI, and TnC isoforms wereassessed by electrophoretic and immunoblot analyses. HU inducedprofound slow-to-fast isoform transitions of all Tn subunits, althoughto different extents and with different time courses. The effectivenessof the isoform transitions was higher for TnT than for TnI and TnC.Indeed, TnI and TnC encompassed minor partial exchanges of slowisoforms with their fast counterparts, whereas the expression patternof fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in thesame manner by HU. This suggests that the slow and fast counterparts ofthe Tn subunit isoforms are regulated independently in response to HU.The changes in TnTf composition occurred in parallel with previouslydemonstrated transitions within the pattern of the fast myosin heavychains in the same muscles.

     

Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein.

Cytosolic glutathione S-transferases were purified from the epithelial cells of human small and large intestine. These preparations were characterized with regard to specific activities, subunit and isoenzyme composition. Isoenzyme composition and specific activity showed little variation from proximal to distal small intestine. Specific activities of hepatic and intestinal enzymes from the same patient were comparable. Hepatic enzymes were mainly composed of 25 kDa subunits. Transferases from small intestine contained 24 and 25 kDa subunits, in variable amounts. Colon enzymes were composed of 24 kDa subunits. In most preparations, however, minor amounts of 27 and 27.5 kDa subunits were detectable. Separation into isoforms by isoelectric focusing revealed striking differences: glutathione S-transferases from liver were mainly basic or neutral, enzymes from small intestine were basic, neutral and acidic, whereas large intestine contained acidic isoforms only. The intestinal acidic transferase most probably was identical with glutathione S-transferase Pi, isolated from human placenta. In the hepatic preparation, this isoform was hardly detectable. The specific activity of glutathione S-transferase showed a sharp fall from small to large intestine. In proximal and distal colon, activity seemed to be about equal. In the ascending colon there might be a relationship between specific activity of glutathione S-transferases and age of the patient, activity decreasing with increasing age.     

Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits

During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (α-subunit) and 65 (β-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10-fold increase in β-subunit and twofold increase in α-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total β-subunit to α-subunit content. The β- to α-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak 1 isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.     

Developmental regulation of type II calcium/calmodulin-dependent kinase isoforms in rat cerebellum

Two distinct isoforms of a Type II calcium/calmodulin-dependent protein kinase were separated from high-speed supernates (cytosol) of rat neonatal [postnatal day 10 (P10)] and adult [postnatal day 40 (P40)] cerebellum using cation-exchange chromatography. The isoenzymes contained variable amounts of three subunits of apparent Mr's of 50 kDa (alpha), 58 kDa (beta'), and 60 kDa (beta). The specific activity of calmodulin-dependent kinase (CaM kinase II) in crude homogenates increased sixfold between P10 and P40 using exogenous MAP 2 as substrate. Cytosol from cerebellum at P40 contained a predominant isoform (approximately 40% of total cytosolic activity) with a 1:5 molar ratio of alpha:beta',beta subunits that eluted with 150 mM NaCl (designated 150) and a less abundant isoform (approximately 20% of total cytosolic activity) containing a 1:8 molar ratio of alpha:beta',beta subunits that eluted with 350 mM NaCl (designated 350). In neonatal cerebellum at P10, the relative abundance of the two isoforms was reversed such that approximately 50% of the cytosolic calmodulin-dependent kinase activity was recovered in the 350 isoform, whereas only 20% of the total cytosolic kinase activity was recovered in the 150 isoform. Previous studies indicate that cerebellar granule cells may contain an all beta',beta isoform of CaM kinase II that lacks alpha subunit. Thus, to assess the cell-specific localization of kinase isoforms within cerebellum, cytosol prepared from primary cultures of rat cerebellar granule cells was applied to cation-exchange chromatography and analyzed for calmodulin-dependent kinase activity. The cells contained both isoforms of the kinase that were present in fresh tissue suggesting that granule cell-enriched cultures express all three kinase subunits. The data demonstrate that rat cerebellum contains unique mixtures of CaM kinase II isoenzymes and that their expression is developmentally regulated.     

Complete primary structure of a newly characterized galactose-specific lectin from the seeds of <Emphasis Type="Italic">Dolichos lablab</Emphasis>

A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively. Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively. The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural conservation. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Analysis of the sucrose synthase gene family in Arabidopsis

The properties and expression patterns of the six isoforms of sucrose synthase in Arabidopsis are described, and their functions are explored through analysis of T-DNA insertion mutants. The isoforms have generally similar kinetic properties. Although there is variation in sensitivity to substrate inhibition by fructose this is unlikely to be of major physiological significance. No two isoforms have the same spatial and temporal expression patterns. Some are highly expressed in specific locations, whereas others are more generally expressed. More than one isoform is expressed in all organs examined. Mutant plants lacking individual isoforms have no obvious growth phenotypes, and are not significantly different from wild-type plants in starch, sugar and cellulose content, seed weight or seed composition under the growth conditions employed. Double mutants lacking the pairs of similar isoforms sus2 and sus3, and sus5 and sus6, are also not significantly different in these respects from wild-type plants. These results are surprising in the light of the marked phenotypes observed when individual isoforms are eliminated in crop plants including pea, maize, potato and cotton. A sus1/sus4 double mutant grows normally in well-aerated conditions, but shows marked growth retardation and accumulation of sugars when roots are subjected to hypoxia. The sucrose synthase activity in roots of this mutant is 3% or less of wild-type activity. Thus under well-aerated conditions sucrose mobilization in the root can proceed almost entirely via invertases without obvious detriment to the plant, but under hypoxia there is a specific requirement for sucrose synthase activity.     

Evidence of active NADP(+) phosphatase in dormant seeds of Avena sativa L

Freshly-harvested seeds of Avena sativa L. do not germinate when imbibed at temperatures higher than 25 degrees C. This high temperature dormancy is due to the seed coats, and to the low activities of glycolysis and the oxidative pentose phosphate pathway (OPP) in the embryo. The analysis by exclusion chromatography of soluble NADP(+) phosphatase activities of embryos revealed two isoforms: a 37 kDa isoform present in both dormant and after-ripened caryopses, and a second isoform, with an apparent molecular weight of 160 kDa, five times more active in embryos of dormant seeds than in the after-ripened ones, after 6 h of imbibition at 30 degrees C. Moreover, the activity of this 160 kDa isoform was three times less in embryos from dormant caryopses when they were grown at 10 degrees C, a permissive temperature for radicle protrusion. These results suggest a correlation between the activity of the 160 kDa NADP(+) phosphatase and the dormancy state of the caryopsis. The two isoforms differed in the pH required for optimal activity: pH 5.7 and 6.5 for the 37 kDa and the 160 kDa phosphatases, respectively. Furthermore, the 160 kDa NADP(+) phosphatase displayed a strong specificity for NADP(+), whereas the 37 kDa isoform was able to hydrolyse numerous other phosphorylated compounds.     

Sucrose phosphate synthase and sucrose accumulation at low temperature

The influence of growth temperature on the free sugar and sucrose phosphate synthase content and activity of spinach (Spinacia oleracea) leaf tissue was studied. When plants were grown at 25°C for 3 weeks and then transferred to a constant 5°C, sucrose, glucose, and fructose accumulated to high levels during a 14-d period. Predawn sugar levels increased from 14- to 20-fold over the levels present at the outset of the low-temperature treatment. Sucrose was the most abundant free sugar before, during, and after exposure to 5°C. Leaf sucrose phosphate synthase activity was significantly increased by the low-temperature treatment, whereas sucrose synthase and invertases were not. Synthesis of the sucrose phosphate synthase subunit was increased during and after low-temperature exposure and paralleled an increase in the steady-state level of the subunit. The increases in sucrose and its primary biosynthetic enzyme, sucrose phosphate synthase, are discussed in relation to adjustment of metabolism to low nonfreezing temperature and freezing stress tolerance.     

The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.     

Investigation of phosphoeno/pyruvate carboxylase (PEPCase) in Mesembryanthemum crystallinum L. in C3 and CAM photosynthetic states

When exposed to osmotic stress, Mesembryanthemum crystallinum plants switch from C₃ to CAM photosynthesis. Phosphoenolpyruvate carboxylase (PEPCase) is a key enzyme in CAM plants, being responsible for the initial fixation of CO₂. In C₃ plants the enzyme has been shown to be involved in the replenishing of TCA cycle intermediates and in the operation of stomatal guard cells. Multiple PEPCase isoforms were observed in C₃-performing leaves with four isoelectric points of 5.2, 5.5, 5.6 and 5.9 and four apparent subunit molecular masses of 105, 108, 113 and 116 kDa. In some instances, subunits of different size possessed exactly the same pI. The induction of CAM led to the predominance of a new isoform of pI 6.5 with subunit molecular mass of 108 kDa, but in addition, changes were observed in some of the isoforms present in the C₃ plant. PEPCase subunits were purified from the C₃ and CAM forms of M. crystallinum and subjected to pep-tide mapping. Two distinct though similar sets of maps were obtained, one from the CAM isoform (pI 6.5) and C₃-associated subunits of pi 5.9 and another for C₃ subunits of pI 5.2 and 5.5. It was inferred from these data that the C₃ isoforms expressed in the leaf were derived from at least two genes. The C₃ isoform (pI 5.9) showing greatest similarity to the CAM isoform in terms of peptide mapping also increased in response to salt stress. It is speculated that the CAM isoform may have evolved from this enzyme.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Occurrence of two isoforms of glutathione reductase in the green alga Chlamydomonas reinhardtii

Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K _m values for the oxidized form of glutathione (approx. 50 M). However, the K _m values for NADPH were different, being 7 M and 28 M for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.Abbreviations GSSG glutathione, oxidized form - GR NAD-PH-glutathione reductase (EC 1.6.4.2) - G3P glyceraldehyde-3-phosphate - pI isoelectric point - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate This work was supported in part by grants NO. PB 87–401, PB 90–99 and BIO 91–1078 of the DGICYT (Ministerio de Educatión y Ciencia, Spain) and the Autonomous Government of Andalusia (Spain). Postdoctoral aid from the Alexander von Humboldt Foundation (Bonn, FRG) to A.S. is also acknowledged.     

Separation,characterization and catalytic properties of Lip2 isoforms from Candida sp. 99-125

Lipase (EC.3.1.1.3) from Candida sp. 99-125 was separated into four isoforms (isoform A, isoform B, isoform C, and isoform D) by two steps of ion exchange chromatography. As analyzed on SDS- and non-denaturing PAGE, the four isoforms were homogenous and had the same molecular weight of approximate 38 kDa. MALDI-TOF peptide mass fingerprinting maps and circular dichroism spectra showed the isoforms had similar peptide patterns belonging to the same protein encoded by the YLlip2 gene and different secondary structures. The isoforms had a little distinct optimum temperature in the range of 20–35 °C, and the same optimum pH (8.0). They remained to be active in methanol, ethanol and ethylene glycol at the concentration of 10% and 20% (v/v) and acetone at the concentration of 10% (v/v), and sensitive to EDTA. Triton X-100, Sodium cholate and CHAPS slightly increased their activities. The metal ion Ca²⁺ and Mg²⁺ had mild effect on lipase activity. The isoforms showed a preference for long chain fatty acid triglyceride (triolein and olive). The lipase purified by one step of ion exchange chromatography and isoforms were less active than crude enzyme to catalyze cetyl alcohol and oleic acid in n-hexane, whereas the presence of small concentration of added water dramatically activated crude lipase but less the purified preparations.     

Knockdown of F1 epsilon subunit decreases mitochondrial content of ATP synthase and leads to accumulation of subunit c

The subunit ε of mitochondrial ATP synthase is the only F₁ subunit without a homolog in bacteria and chloroplasts and represents the least characterized F₁ subunit of the mammalian enzyme. Silencing of the ATP5E gene in HEK293 cells resulted in downregulation of the activity and content of the mitochondrial ATP synthase complex and of ADP-stimulated respiration to approximately 40% of the control. The decreased content of the ε subunit was paralleled by a decrease in the F₁ subunits α and β and in the F_o subunits a and d while the content of the subunit c was not affected. The subunit c was present in the full-size ATP synthase complex and in subcomplexes of 200–400 kDa that neither contained the F₁ subunits, nor the F_o subunits. The results indicate that the ε subunit is essential for the assembly of F₁ and plays an important role in the incorporation of the hydrophobic subunit c into the F₁-c oligomer rotor of the mitochondrial ATP synthase complex.     

Identification of F0 subunits in the rat liver mitochondrial F0F1-ATP synthase.

In order to identify the subunits constituting the rat liver F0F1-ATP synthase, the complex prepared by selective extraction from the mitochondrial membranes with a detergent followed by purification on a sucrose gradient has been compared to that obtained by immunoprecipitation with an anti-F1 serum. The subunits present in both preparations that are assumed to be authentic components of the complex have been identified. The results show that the total rat liver F0F1-ATP synthase contains at least 13 different proteins, seven of which can be attributed to F0. The following F0 subunits have been identified: the subunit b (migrating as a 24 kDa band in SDS-PAGE), the oligomycin-sensitivity-conferring protein (20 kDa), and F6 (9 kDa) that have N-terminal sequences homologous to the beef-heart ones; the mtDNA encoded subunits 6 (20 kDa) and 8 (less than 7 kDa) that can be synthesized in isolated mitochondria; an additional 20 kDa protein that could be equivalent to the beef heart subunit d.