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.     

Expression of Na,K-ATPase isoforms in human heart.

The expression pattern of the multiple isoforms of Na,K-ATPase was examined in the human heart. Isoform specific oligonucleotide probes for the alpha 1, alpha 2, alpha 3 and beta 1 subunits were used to probe Northern blots. The adult human ventricle expresses mRNAs for all three alpha subunit isoforms in addition to beta 1 subunit mRNA.     

大鼠肌肉神经白介素纯化和生化特性及其与磷酸葡萄糖异位酶的关系

通过硫酸铵沉淀、DE_(52)层析、超滤和高压羟基磷灰石层析,从大鼠肌肉条件培养液中得到电泳纯的神经白介素(NLK)。未发现NLK的神经营养活性(维持鸡胚背根神经元存活和促进脊髓神经元突起伸展)及免疫活性(促进外周血单个核细胞产生免疫球蛋白),却有较强的6-磷酸葡萄糖异位酶(GPI)活性。SDS-PAGE测得亚基分子量为56kD;IEF示五条细而相距很近的带,PⅠ值分别为8.20,8.15,8.10,7.90,7.75。Western Blot证实,PAGE和IEF的五条带均可与抗GPI抗体结合,即五种同工酶形式;NLK与GPI的氨基酸组成相近,BrCN水解NLK和GPI得剖相同的肽谱。许多研究表明:NLK不作为一种神经营养因子,而可能是糖酵解酶,即6-磷酸葡萄糖异位酶。     

Multiple forms of ADP‐glucose pyrophosphorylase from tomato leaf

ADP‐glucose pyrophosphorylase (AGP, EC 2.7.7.27) was partially purified from tomato ( Lycopersicon esculentum Mill. cv. Laura) leaf by a procedure previously used for purification of AGP from tomato pericarp. SDS‐PAGE and western blot analysis of the final preparation indicated that the leaf enzyme is composed of two subunits of 50 and 54 kDa. Two‐dimensional PAGE and western blot analysis of the same preparation, however, revealed at least six isoforms of the large subunit and two isoforms of the small subunit. The leaf AGP is very sensitive to regulation by 3‐phosphoglycerate and inorganic phosphate. Its properties are compared to those of AGP from tomato fruit.     

Purification and characterization of multiple species (isolectins) of a slime mold lectin implicated in intercellular adhesion.

An improved purification procedure for the carbohydrate-binding proteins (lectins) of cohesive Polysphondylium pallidum cells has been devised. The procedure uses extraction of cells with lactose-containing buffer followed by ammonium sulfate precipitation and affinity chromatography of the redissolved precipitate on a column of acid-treated Sepharose 6B. All hemagglutination activity is adsorbed to the column and recoveries are about 70% of the activity of the starting cell lysate. Sodium dodecyl sulfate-gel electrophoresis of the protein obtained with this procedure resolved three subunits with molecular weights of 26,500 (A), 26,000 (B), and 25,000 (C). Three species are resolved by isoelectric focusing with apparent pI values of 6.4 (I), 7.3 (II), and 7.5 (III) which contain Subunits A, B, and C in the following ratios: I, B:C at 2:1; II, A:B at 2:1, and III, A:B at 1:2. All three isoforms agglutinate rabbit and human type O erythrocytes and are thus isolectins. Isoforms II and III are separated from Isoform I by galactose-gradient elution of the Sepharose 6B column. Isoforms II and III aggregate extensively (nonamers and multiples thereof), but reduction with 2-mercaptoethanol reverses this process yielding a single species of Mr = 73,000 (trimer). Isoform I exists as trimers and hexamers and reduction has no effect on this distribution. Amino acid compositions and tryptic peptide maps of S-[14C]carboxymethyl-isolectins indicate that Subunits A and B are very similar and may represent the same peptide chain, while Subunit C is a peptide quite distinct from A and B.     

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.     

Separation of glutathione transferase subunits from Proteus vulgaris by two-dimensional gel electrophoresis

Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.     

Acetyl Coenzyme A Synthetase (ADP Forming) from the Hyperthermophilic Archaeon Pyrococcus furiosus: Identification, Cloning, Separate Expression of the Encoding Genes, acdAI and acdBI, in Escherichia coli, and In Vitro Reconstitution of the Active Heterotetrameric Enzyme from Its Recombinant Subunits

Acetyl-coenzyme A (acetyl-CoA) synthetase (ADP forming) represents a novel enzyme in archaea of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) --> acetate + ATP + CoA). Two isoforms of the enzyme have been purified from the hyperthermophile Pyrococcus furiosus. Isoform I is a heterotetramer (alpha(2)beta(2)) with an apparent molecular mass of 145 kDa, composed of two subunits, alpha and beta, with apparent molecular masses of 47 and 25 kDa, respectively. By using N-terminal amino acid sequences of both subunits, the encoding genes, designated acdAI and acdBI, were identified in the genome of P. furiosus. The genes were separately overexpressed in Escherichia coli, and the recombinant subunits were reconstituted in vitro to the active heterotetrameric enzyme. The purified recombinant enzyme showed molecular and catalytical properties very similar to those shown by acetyl-CoA synthetase (ADP forming) purified from P. furiosus.     

Purification and properties of a beta-galactosidase from carambola fruit with significant activity towards cell wall polysaccharides

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.     

A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex).

Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.     

Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase (GPI) deficiency

Glucose-6-phosphate isomerase (GPI) deficiency, an autosomal recessive genetic disorder with the typical manifestation of nonspherocytic haemolytic anaemia, can be associated in some cases with neurological impairment. GPI has been found to be identical to neuroleukin (NLK), which has neurotrophic and lymphokine properties. To focus on the possible effects of GPI mutations on the central nervous system through an effect on neuroleukin activity, we analysed DNA isolated from two patients with severe GPI deficiency, one of them with additional neurological deficits, and their families. The neurologically affected patient (GPI Homburg) is compound heterozygous for a 59 A→C (H20P) and a 1016 T→C (L339P) exchange. Owing to the insertion of proline, the H20P and L339P mutations are likely to affect the folding and activity of the enzyme. In the second family studied, the two affected siblings showed no neurological symptoms. The identified mutations are 1166 A→G (H389R) and 1549 C→G (L517V), which are located at the subunit interface. We propose that mutations that lead to incorrect folding destroy both catalytic (GPI) and neurotrophic (NLK) activities, thereby leading to the observed clinical symptoms (GPI Homburg). Those alterations at the active site, however, that allow correct folding retain the neurotrophic properties of the molecule (GPI Calden).     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.     

Characterization of two sucrose synthase isoforms in sugarbeet root

Two sucrose synthase isoforms (EC 2.4.1.13) have been identified in developing sugarbeet (Beta vulgaris L.) roots. To aid in understanding the physiological significance of these multiple sucrose synthase isoforms, the two isoforms were partially purified and some of their physical and kinetic properties determined. Both isoforms were tetrameric proteins with native molecular masses of 320 kDa. The isoforms exhibited similar kinetic properties as well as similar changes in activity in response to changes in temperature. The isoforms differed, however, in their subunit composition. Sucrose synthase isoform I (SuSyI) was composed of two 84 kDa subunits and two 86 kDa subunits. Sucrose synthase isoform II (SuSyII) was a homotetramer with a subunit size of 86 kDa. The amino acid composition of the two subunits was similar, although differences in alanine, glycine, isoleucine and lysine content were noted. The activity of the two isoforms differed in response to varying pH conditions. The optimum pH for sucrose cleaving activity was observed at pH 6.0 and 6.5 for SuSyI and SuSyII, respectively. The optimum pH for sucrose synthesizing activity occurred at pH 7.5 and 7.0 for SuSyI and SuSyII, respectively. The observed differences in subunit composition and reactivity at different pH values suggest that multiple isoforms of sucrose synthase may provide a mechanism to regulate sucrose metabolism in sugarbeet root by differential regulation of expression of the two isoforms and modulation of their activity by changes in cellular pH.     

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.     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Purification and characterization of interleukin 1 receptor level antagonist proteins from THP-1 cells

Culture medium conditioned by phorbol 12-myristate 13-acetate-differentiated THP-1 cells contained interleukin 1 (IL-1) antagonist activity as measured by inhibition of both IL-1 beta binding to receptors on YT cells and inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by LBRM-33-1A5 T cells. Based on their ability to compete for 125I-IL-1 beta binding to receptors on YT cells, four distinct antagonist proteins were purified from THP-1 cell conditioned medium using a combination of ion-exchange, hydrophobic interaction, and size exclusion chromatographies. The four proteins had different isoelectric points with molecular masses in the range 22-26 kDa and had similar specific activities for inhibition of IL-1 beta binding to cell surface receptors (Ki values 0.33-0.64 nM) and for inhibition of IL-1/phytohemagglutinin-stimulated IL-2 synthesis by 1A5 cells (IC50 values 25-100 pM). Amino-terminal sequence analysis of the two major forms (25 kDa/pI 5.1 and 22 kDa/pI 5.8) revealed complete identity for the first 27 residues in both forms. Based on the results of peptide mapping, amino acid compositional analysis and immune blotting, all of the forms were deduced to be variants of a common protein. Deglycosylation of the antagonist proteins with N-glycanase converted them to a common form (22 kDa/pI 5.8), indicating that the four isoforms represent glycosylation variants of a common protein and that asparagine-linked oligosaccharides are responsible for the observed size and charge heterogeneity.     

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.     

Separation of Glutathione Transferase Subunits from <Emphasis Type="Italic">Proteus vulgaris</Emphasis> by Two-Dimensional Gel Electrophoresis

Cytosolic glutathione transferases of Proteus vulgaris were purified by affinity chromatography and characterized by two-dimensional gel electrophoresis. Four different subunits were identified, and each subunit contained a different molecular mass, ranging from 26.2 kDa to 28.5 kDa; a different pI value, ranging from 8.2 to 9.4; and a different amount of protein fraction, ranging from 10% to 56%. All four subunits existed as basic proteins (pI > 7.0). From these results, we concluded that multiple forms of glutathione transferase enzymes existed in Proteus vulgaris, and four different glutathione transferase subunits were separated by 2-D gel electrophoresis.Received 23 September 2002 / Accepted 19 December 2002     

Identification of nuclear factor IV/Ku autoantigen in a human 2D-gel protein database. Modification of the large subunit depends on cellular proliferation

Nuclear Factor IV (NFIV) is a heterodimeric DNA-binding protein from HeLa cells, recognizing molecular ends and is identical to the autoantigenic target Ku. We have identified the two NFIV/Ku subunits, by comigration, in the 2D-gel database of transformed human amnion cell (AMA) proteins. We observed that the large subunit of NFIV/Ku consists of at least 3 charge variants that correspond to SSP IEFs 5705 (81.2 kDa, pI 5.74), 6707 (81.2 kDa, pI 5.67) and 6706 (81.9 kDa, pI 5.60) in the AMA catalogue. The relative amounts of the 2 major variants (IEFs 5705 and 6707) was dependent on the state of cell proliferation. Inhibition of DNA-synthesis by hydroxyurea also changed the relative levels of the variants, whereas aphidicolin or a thymidine block had no effect. These results suggest a possible role for NFIV/Ku in DNA replication.     

Tissue specificity of E subunit isoforms of plant vacuolar H(+)-ATPase and existence of isotype enzymes

Immunoblot analyses and partial amino acid sequencings revealed that both the 40- (E1) and 37-kDa (E2) subunits of V-ATPase in the pea epicotyl were E subunit isoforms. Similarly, both the 35- (D1) and 29-kDa (D2) subunits were D subunit isoforms, although the similarity of the amino acid sequences is still unknown. In immunoblot analyses, two or three E subunit isoforms with molecular masses ranging from 29 to 40 kDa were detected in other plants. Two isotypes of V-ATPase from the pea epicotyl were separated by ion exchange chromatography and had subunit compositions differing only in the ratio of E1 and E2. There was a difference in the V(max) and K(m) of ATP hydrolysis between the two isotypes. E1 was scarcely detected in crude membrane fractions from the leaf and cotyledon, while E2 was detected in fractions from all of the tissues examined. The compositions of D subunit isoforms in the leaf and epicotyl were different, and the vacuolar membrane in the leaf did not contain D2. The efficiency of H(+) pumping activity in the vacuolar membrane of the leaf was higher than that of the epicotyl. The results suggest that the presence of the isoforms of D and E subunits is characteristic to plants and that the isoforms are closely related to the enzymatic properties.