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1.
Biosynthesis of isocitrate lyase, a tetrameric enzyme of the glyoxysomal matrix, was studied in Neurospora crassa, in which the formation of glyoxysomes was induced by a substitution of sucrose medium by acetate medium. 1. Translation of Neurospora mRNA in reticulocyte lysates yields a product which has the same apparent molecular weight as the subunit of the functional enzyme. Using N-formyl[35S]methionyl-tRNAfMet as a label, the translation product shows the same apparent size which indicates that the amino terminus has no additional "signal'-type sequence. 2. Read-out systems employing free and membrane-bound polysomes show that only free ribosomes are active in the synthesis of isocitrate lyase. 3. Isocitrate lyase synthesized in reticulocyte lysate is released into the supernatant and is soluble in a monomeric form. It interacts with Triton X-100 to form mixed micells in contrast to the functional tetrameric form. 4. Transfer of isocitrate lyase synthesized in vitro into isolated glyoxysomes is suggested by results of experiments in which supernatants from reticulocyte lysates are incubated with a particle fraction isolated from acetate-grown cells. No transfer occurs when particles from non-induced cells are employed. Resistance to added proteinase is used as a criterion for transmembrane transfer. The data support a post-translational transfer mechanism for isocitrate lyase. They suggest that isocitrate lyase passes through a cytosolic precursor pool as a monomer and is transferred into glyoxysomes. 相似文献
2.
The in vitro phosphorylation of isocitrate lyase was demonstrated in partially purified sonic extracts ofEscherichia coli. Extracts were incubated with [gamma32P]-ATP and subsequently analyzed by two-dimensional polyacrylamide gel electrophoresis. Isocitrate lyase was determined to be phosphorylated by autoradiography and Western blot analyses of the gels. Purified isocitrate lyase comigrates with the phosphorylated form of the enzyme; this suggests that the enzyme may become catalytically active concomitant with phosphorylation. 相似文献
3.
Ruchti, M. and Widmer, F. 1986. Isocitrate lyase from germinatingsoybean cotyledons: purification and characterization.J.exp. Bot. 37: 16851690. Isocitrate lyase (E.C. 4.1.3.1
[EC]
) was purified from the cotyledonsof 7-d-old soybean seedlings. Three molecular forms were detectedwith pi values of 6·46, 6·25 and 6·0. Themain form (pl = 6·46) had an approximate Mr of 130000,a pH optimum of 8·0, a Km (isocitrate) close to 2·0mol m3 and a molecular activity of 615 min 1 at25 °C. The purified enzyme is not a glycoprotein and isheat labile. Key words: Isocitrate lyase, soybean 相似文献
4.
A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds 总被引:1,自引:1,他引:0
Prof. Peter J. Gruber Richard N. Trelease Wayne M. Becker Eldon H. Newcomb 《Planta》1970,93(4):269-288
Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles. 相似文献
5.
The glyoxylate cycle enzyme, isocitrate lyase (EC 4.1.3.1) was purified from cotyledons of Citrullus vulgaris (watermelon). The final preparation, which had been 97-fold purified with a specific activity of 16.1 units/mg protein in a yield of 36%, was homogeneous by gel- and immunoelectrophoretic criteria. The tetrameric enzyme had: a molecular weight of 277 000, a sedimentation coefficient of 12.4 s, and a Km for Ds-isocitrate equal to 0.25 mM. Isocitrate lyase from this source is not a glycoprotein as shown by total carbohydrate content after precipitation by trichloroacetic acid of the purified enzyme. Reduction of the enzyme with thiols increased activity and maximal activity was obtained with at least 5 mM dithiothreitol. EDTA partially substituted for thiol in freshly isolated enzyme. Watermelon isocitrate lyase was also protected against thermal denaturation at 60° for at least 1 hr by 5 mM Mg2+ plus 5 mM oxalate. Oxalate was a competitive inhibitor with respect to isocitrate (Ki: 1.5 μM, pH 7.5, 30°). 相似文献
6.
Vasily N. Popov Abir U. Igamberdiev Claus Schnarrenberger Sergei V. Volvenkin 《FEBS letters》1996,390(3):258-260
The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, have been detected in liver of foodstarved rats. Activities became measurable 3 days and peaked 5 days after the beginning of starvation. Both enzymes were found in the peroxisomal cell fraction after organelle fractionation by isopycnic centrifugation. Isocitrate lyase was purified 112-fold by ammonium sulfate precipitation, and chromotography on DEAE-cellulose and Toyopearl HW-65. The specific activity of the purified enzyme was 9.0 units per mg protein. The Km(isocitrate) was 68 μM and the pH optimum was at pH 7.4. Malate synthase was enriched 4-fold by ammonium sulfate precipitation. The enzyme had a Km(acetyl-CoA) of 0.2 μM, a Km(glyoxylate) of 3 mM and a pH optimum of 7.6. 相似文献
7.
Summary After the functional transition of glyoxysomes to leaf peroxisomes during the greening of pumpkin cotyledons, the reverse microbody transition of leaf peroxisomes to glyoxysomes occurs during senescence. Immunocytochemical labeling with protein A-gold was performed to analyze the reverse microbody transition using antibodies against a leaf-peroxisomal enzyme, glycolate oxidase, and against two glyoxysomal enzymes, namely, malate synthase and isocitrate lyase. The intensity of labeling for glycolate oxidase decreased in the microbodies during senescence whereas in the case of malate synthase and isocitrate lyase intensities increased strikingly. Double labeling experiments with protein A-gold particles of different sizes showed that the leaf-peroxisomal enzymes and the glyoxysomal enzymes coexist in the microbodies of senescing pumpkin cotyledons, indicating that leaf peroxisomes are directly transformed to glyoxysomes during senescence. 相似文献
8.
Phosphorylation of isocitrate lyase in Escherichia coli 总被引:2,自引:0,他引:2
Isocitrate lyase from Escherichia coli becomes phosphorylated in vitro by an endogenous kinase when partially purified extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with histidine modifying reagents, and alkaline hydrolysis of in vitro phosphorylated enzyme indicated the presence of a phosphohistidine residue. Phosphorylation of isocitrate lyase can also occur in vivo, which indicates a possible regulatory significance of this modification. In addition to phosphorylation, isocitrate lyase is capable of incorporating label from both [alpha-32P]ATP and [14C]ATP suggesting that more than one type of covalent modification occurs on this enzyme. This report reviews the studies which have demonstrated the phosphorylation and modification of isocitrate lyase from Escherichia coli. 相似文献
9.
Bertold Hock 《Planta》1969,85(4):340-350
Summary The isocitrate lyase activity (E.C. 4.1.3.1.) from watermelon cotyledons (Citrullus vulgaris
Schrad.) is inhibited by white light (Fig. 5). To exclude artefacts during enzyme preparation the following experiments were performed (Table 1 and 2): 1. Mixing of raw extracts from cotyledons of light and dark grown seedlings. 2. Joint homogenization a) of cotyledons from light and dark grown seedlings, b) of purified isocitrate lyase together with cotyledons from light or dark grown seedlings. The total activity corresponded to an amount which was expected for the sum of the individual activities. The results justify the conclusion that the inhibition of the isocitrate lyase by light is real, and that the measured enzyme activities are close to the true enzyme concentrations in the plant tissue. — The relatively slow inhibition of the enzyme activity caused by light seems to be correlated with the formation of the photosynthetic apparatus. 相似文献
10.
F R Khan M Saleemuddin M Siddiqi B A McFadden 《Archives of biochemistry and biophysics》1977,183(1):13-23
Isocitrate lyase has been purified from flax (Linum usitatissimum) seedlings. The final preparation was homogeneous by the criteria of polyacrylamide disc gel electrophoresis, immunodiffusion, and immunoelectrophoresis. From exclusion chromatography on Sephadex G-200, the molecular weight and Stoke's radius of the enzyme were 264,000 and 5.28 × 10?7 cm, respectively. The subunit molecular weight was 67,000. Thus, the enzyme appears to be tetrameric. The enzyme required Mg2+ and cysteine for activity. The optimal pH of the enzyme was 7.5 both in Tris and in phosphate buffers. There are three disulfide bridges and two of eight cysteine residues are buried. Inactivation of isocitrate lyase resulted from short-term modification of enzymatic thiols but this could be reversed by added thiols. The enzyme was competitively inhibited by glyoxylate, l-tartrate, and malonate in catalysis of isocitrate cleavage. 相似文献
11.
Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation. 总被引:1,自引:0,他引:1
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The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA. 相似文献
12.
Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase 总被引:10,自引:0,他引:10
E M Weir H Riezman J M Grienenberger W M Becker C J Leaver 《European journal of biochemistry》1980,112(3):469-477
The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light. 相似文献
13.
W K?ller J Frevert H Kindl 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(2):167-176
1) Albumins and globulins were prepared from dry seeds of cucumbers (Cucumis sativus) by differential extraction. The globulin fraction was analyzed by gel electrophoresis under denaturing conditions in the presence and absence of mercaptoethanol. The subunit (Mr = 54000) of the tetramer (Mr = 240000) was shown to be composed of two different peptides. Microheterogeneity rendered the exact interpretation of the analysis difficult. 2) Glyoxysomal proteins were already present in dry seeds: malate synthase, isocitrate lyase, citrate synthase, malate dehydrogenase, catalase and crotonase could be detected unequivocally. It was demonstrated that the enzymatic and immunological properties of malate synthase and isocitrate lyase were not distinguishable from that of enzymes assigned to glyoxysomes of fully developed cotyledons. 3) Homogenates prepared from seeds by cautious cell disintegration were subjected to sucrose density gradient centrifugation and yielded microbody and protein body fractions, among other things. 相似文献
14.
The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle. 相似文献
15.
《Plant science》1986,46(2):97-101
Isocitrate lyase (ICL) was highly purified from cotyledons of dark-grown pumpkin (Cucubita maxima Kuri Nankin) seedlings by rapid means, and a method was devised to determine glyoxylate using the enzyme in combination with yeast isocitrate dehydrogenase (NADP+). The enzymic properties of both enzymes were adequate for determination of varying concentrations of glyoxylate. The intracellular content of glyoxylate in Euglena gracilis Klebs strain Z Pringsheim growing photosynthetically in air was determined to be 2.0 nmol mg−1 chlorophyll using this method. 相似文献
16.
The glyoxysomal enzymes isocitrate lyase and catalase have been isolated from etiolated cucumber (Cucumis sativus) cotyledons. The enzymes co-purified through polyethyleneimine precipitation and (NH4)2SO4 precipitation, and were resolved by gel filtration on Sepharose 6B followed by chromatography on diethylaminoethyl-cellulose (isocitrate lyase) or hydroxylapatite (catalase). Purity of the isolated enzymes was assessed by sodium dodecyl sulfate-polyacrylamide electrophoresis, isoelectric focusing, and immunoelectrophoresis. Antibodies raised to both enzymes in rabbits and in tumor-bearing mice were shown to be monospecific by immunoelectrophoresis against total homogenate protein. Isocitrate lyase and catalase represent about 0.56% and 0.1%, respectively, of total extractable cotyledonary protein. Both enzymes appear to be present in a single form. Molecular weights of the native enzymes and its subunits are 225,000 and 54,500 for catalase, and 325,000 and 63,500 for isocitrate lyase. The pH optimum for isocitrate lyase is about 6.75 in morpholinopropane sulfonic acid buffer, but varies significantly with buffer used. The Km for d-isocitrate is 39 micromolar. A double antibody technique (rabbit anti-isocitrate lyase followed by 125I-labeled goat anti-rabbit immunoglobulin G) has been used to visualize isocitrate lyase subunit protein on sodium dodecyl sulfate-polyacrylamide with high specificity and sensitivity. 相似文献
17.
Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (Ki = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 +/- 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer). 相似文献
18.
Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme. 总被引:1,自引:1,他引:0
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Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate. 相似文献
19.
Gibberellic acid (GA3) stimulates isocitrate lyase activity of the endosperm during germination of castor bean seeds. Isocitrate lyase from castor bean was purified and an antibody to it was prepared from rabbit serum. This antibody was used to measure the amounts of isocitrate lyase-mRNA using an in vitro translation system. No specific stimulation of isocitrate lyase-mRNA by application of GA3 was detected. The stimulation of isocitrate lyase activity by exogenous GA3 may be accounted for by the action of the growth substance in advancing the overall production of rRNA and mRNA which accelerates the rate of total protein synthesis during germination. The application of Amo 1618 retards the production of isocitrate lyase activity but also retards protein synthesis in general. This suggests that endogenous gibberellins also act non-specifically in the regulation of protein synthesis during castor bean germination.Abbreviations SDS
sodium dodecyl sulphate
- GA3
gibberellic acid
- PAGE
polyacrylamide gel electrophoresis 相似文献
20.
KOUKI ONO MASAHIRO OKIHASHI HIROSHI INUI KAZUTAKA MIYATAKE SHOZABURO KITAOKA YOSHIHISA NAKANO 《The Journal of eukaryotic microbiology》1994,41(6):536-539
Isocitrate lyase was purified to homogeneity from ethanol-grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS-polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The Km values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis. 相似文献