On the significance of the prosthetic group composition of citrate lyase.

1. Klebsiella aerogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase. 2. The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes. 3. As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes. 4. Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes. 5. The prosthetic group of citrate lyase, phosphoribosyl dephospho-CoA, apparently represents no incidental, phosphopantetheine-replacing aberration. The requirement of citrate lyase for the CoA-like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.     

The enzyme complex citramalate lyase from Clostridium tetanomorphum.

1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.     

The prosthetic group of citrate-lyase acyl-carrier protein.

The acyl carrier protein of citrate lyase contains adenine, phosphate, sugar, cysteamine, beta-alanine and pantoic acid in a molar ratio of 1:2:2:1:1:1. Peptides containing these components in the same stoichiometric relationship were isolated after proteolytic digestion of acyl carrier protein. All components were linked together in a single prosthetic group. This was released from the peptide by mild alkaline hydrolysis. Under these conditions a phosphodiester bond is cleaved which links the prosthetic group to a serine residue of the peptide. Incubation of the prosthetic-group-containing peptide with phosphodiesterase I yielded 4'-phosphopantetheine and adenylic acid. The 5'-AMP was not free but was substituted by presumably an acidic sugar residue, which was released by mild acid hydrolysis yielding free 5'-AMP. It was concluded from these results that the prosthetic group of citrate lyase acyl carrier protein consists of a substituted isomeric dephospho-CoA. This is bound to the protein by the 5'-phosphate group of adenylic acid. The 4'-phosphopantetheine residue is bound by a phosphodiester linkage to the 2' or 3' position of ribose and the remaining hydroxyl group of ribose is substituted with presumably an acidic sugar residue. The structural similarities of this prothetic group and coenzyme A are discussed and related to the catalytic properties of citrate lyase.     

Citrate lyase from Klebsiella pneumoniae. The complete primary structure of the acyl lyase subunit

The primary structure of the beta-subunit (acyl lyase subunit) of citrate lyase from Klebsiella pneumoniae (ATCC 13,882) was determined with protein chemical methods. The polypeptide chain consists of 289 amino acid residues and has a molecular mass of 31,352 Da. The two half-cystine residues of the subunit are present as cysteines and not involved in disulfide bridges. The sequence shows no homology to known sequences of proteins or nucleic acids and reads (sequence; see text)     

Incorporation of pantothenate into citrate lyase by a pantothenateless mutant of Klebsiella pneumoniae.

A pantothenate-requiring mutant of Klebsiella pneumoniae was isolated. The mutant showed an absolute dependence on pantothenate for growth. When grown in the presence of [14C]pantothenate, the mutant incorporated [14C]pantothenate into citrate lyase (3.4 mol/mol of enzyme). Analysis of a double-labeled enzyme ([14C]pantothenate and [3H]acetate) by gel electrophoresis in sodium dodecyl sulfate showed that both 3H and 14C were associated solely with the smallest subunit, the acyl carrier protein of citrate lyase.     

On the mechanism of action of isocitrate lyase.

1. The enzymes citrate lyase and isocitrate lyase catalyse similar reactions in the cleavage of citrate to acetate plus oxaloacetate and of isocitrate to succinate plus glyoxylate, respectively. 2. Nevertheless, the mechanism of action of each enzyme appears to be different from each other. Citrate lyase is an acyl carrier protein-containing enzyme complex whereas isocitrate lyase is not. The active form of citrate lyase is an acetyl-S-enzyme but that of isocitrate lyase is not a corresponding succinyl-S-enzyme. 3. In contrast to citrate lyase, the isocitrate enzyme is not inhibited by hydroxylamine nor does it acquire label if treated with appropriately labelled radioactive substrate. 4. Isotopic exchange experiments performed in H18-2O with isocitrate as a substrate produced no labelling in the product succinate. This was shown by mass-spectrometric analysis. 5. The conclusion drawn from these results is that no activation of succinate takes place on the enzyme through transient formation of succinic anhydride or a covalently-linked succinyl-enzyme, derived from this anhydride.     

抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。     

2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans: characterization and comparison of both enzymes.

In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources ( approximately 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mM pyruvate, 150 mM succinate and 10 microM PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.     

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).     

Characterization of citrate lyase from Clostridium sporosphaeroides

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of l-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in star form.     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997     

Klebsiella pneumoniae genes for citrate lyase and citrate lyase ligase: localization, sequencing, and expression

In the course of studies on anaerobic citrate metabolism in Klebsiella pneumoniae, the DNA region upstream of the gene for the sodium-dependent citrate carrier (dtS) was investigated. Nucleotide sequence analysis revealed a cluster of five new genes that were oriented inversely to citS and probaby form an operon. The genes were named citCDEFG. Based on known protein sequence data, the gene products derived from citD, citE and citF could be identified as the λ-, β-, and α-subunits of citrate lyase, respectively. This enzyme catalyses the cleavage of citrate to oxaloacetate and acetate. The gene product derived from citC (calculated M_r 36476) exhibited no obvious similarity to other proteins. In the presence of acetate and ATP, cell extracts from a citC-expressing Escherichia coli strain were able to reactivate purified citrate lyase from K. pneumoniae that had been inactivated by chemical deacetylation of the prosthetic group. This represents 5-phosphoribosyi-dephospho-acetyl-coenzyme A which is covalently bound to serine-14 of the acyl carrier protein (λ-subunit). CitC was thus identified as acetate:SH-citrate lyase ligase. The function of the gene product derived from citG (M_r 32 645) has not yet been identified. Expression of the CitCDEFG gene cluster in E. coli led to the formation of citrate lyase which was active only in the presence of acetyl-coenzyme A, a compound known to substitute for the prosthetic group. These and other data strongly indicated that the enzyme synthesized in E. coli lacked its prosthetic group. Thus, additional genes besides citCDEFG appear to be required for the formation of holo-citrate lyase.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

Biosynthesis of the prosthetic group of citrate lyase

Citrate lyase (EC 4.1.3.6) catalyzes the cleavage of citrate to acetate and oxaloacetate and is composed of three subunits (alpha, beta, and gamma). The gamma-subunit serves as an acyl carrier protein (ACP) and contains the prosthetic group 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA, which is attached via a phosphodiester linkage to serine-14 in the enzyme from Klebsiella pneumoniae. In this work, we demonstrate by genetic and biochemical studies with citrate lyase of Escherichia coli and K. pneumoniae that the conversion of apo-ACP into holo-ACP is dependent on the two proteins, CitX (20 kDa) and CitG (33 kDa). In the absence of CitX, only apo-ACP was synthesized in vivo, whereas in the absence of CitG, an adenylylated ACP was produced, with the AMP residue attached to serine-14. The adenylyltransferase activity of CitX could be verified in vitro with purified CitX and apo-ACP plus ATP as substrates. Besides ATP, CTP, GTP, and UTP also served as nucleotidyl donors in vitro, showing that CitX functions as a nucleotidyltransferase. The conversion of apo-ACP into holo-ACP was achieved in vitro by incubation of apo-ACP with CitX, CitG, ATP, and dephospho-CoA. ATP could not be substituted with GTP, CTP, UTP, ADP, or AMP. In the absence of CitG or dephospho-CoA, AMP-ACP was formed. Remarkably, it was not possible to further convert AMP-ACP to holo-ACP by subsequent incubation with CitG and dephospho-CoA. This demonstrates that AMP-ACP is not an intermediate during the conversion of apo- into holo-ACP, but results from a side activity of CitX that becomes effective in the absence of its natural substrate. Our results indicate that holo-ACP formation proceeds as follows. First, a prosthetic group precursor [presumably 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA] is formed from ATP and dephospho-CoA in a reaction catalyzed by CitG. Second, holo-ACP is formed from apo-ACP and the prosthetic group precursor in a reaction catalyzed by CitX.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Substrate specificity of citrate lyase deacetylase of Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris.

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas palustris was investigated with regard to its kinetic properties and its subunit composition. This enzyme was inactivated by citrate lyase deacetylase (EC 3.1.2.-) of Rhodopseudomonas gelatinosa. A corresponding cross-reaction was measured with partially purified deacetylase of R. palustris and citrate lyase of R. gelatinosa. The three different subunit types (alpha, beta, and gamma) of citrate lyase from R. gelatinosa wee purified to homogeneity, and antibodies were prepared against each of the three subunits and against the native enzyme complex. In corresondence with the enzymatic interactions, immunological cross-reactions were found between anti-enzyme and anti-large subunit antibodies and citrate lyase from R. palustris. On the other hand, no immunological cross-reactions were detectable among each of the antibodies and citrate lyases from Enterobacter aerogenes, Streptococcus diacetilactis, and Clostridium sphenoides. Antibodies against the large subunit of citrate lyase inhibited the deacetylase, but antibodies against the middle and small subunits did not, indicating that the large subunits of citrate lyase are involved in binding the deacetylase.     

Characterization of antibodies to chicken riboflavin carrier protein: Antigenicity of the tryptic fragments

The antigenecity of tryptic fragments of reduced and carboxymethylated chicken riboflavin carrier protein were studied. The tryptic sites of the native riboflavin carrier protein bound to riboflavin were inaccessible. The molecular weight and the elution profile on high performance liquid chromatography (TSK 545 DEAE) were unaltered at an enzyme to substrate ratio of 1:31. However, carboxymethylated riboflavin carrier protein could be cleaved into 3 or 4 fragments at an enzyme to substrate ratio of 1:250 or 1:125. Chromatographic separation of the tryptic fragments on high pressure liquid chromatography (TSK 545 DEAE) revealed the presence of two fragments with different elution profiles but similar molecular weight 26 ±2 kDa. Only one fragment (associated with peak 2) had the ability to displace chicken riboflavin carrier protein in an homologous chicken riboflavin carrier protein radioimmunoassay. Thus, carboxymethylated ribotlavin carrier protein which does not compete with chicken riboflavin carrier protein in the radioimmunoassay, on mild trypsinization generates a fragment which interacts with chicken riboflavin carrier protein in radioimmunoassay.     

The distinctiveness of ATP:citrate lyase from Aspergillus nidulans

ATP:citrate lyase (ACL), an important enzyme in lipid synthesis, has been purified from Aspergillus nidulans to a specific activity of 19.6 micromol min(-1) mg(-1), almost twice that of any other purified ACL and shown to be distinct from any previously purified ACL. The enzyme is a 371+/-31 kDa hexamer of 3 alpha, 3 beta proteins, unlike the 4 alpha tetramer found in rats or yeasts. The molecular weights of the alpha and beta protein subunits were determined by SDS-PAGE to be 70 and 55 kDa.ACL in A. nidulans (unlike Aspergillus niger) appears to be regulated by the carbon source present in the media. In crude extracts, it was found at high activity (88 micromol min(-1) mg protein(-1)) in glucose-grown cells but only at low activity (10 micromol min(-1) mg protein(-1)) in acetate-grown cells.     

A novel enzyme, citryl-CoA lyase, catalysing the second step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6

A novel enzyme catalysing citryl-CoA cleavage to acetyl-CoA and oxaloacetate was purified from Hydrogenobacter thermophilus TK-6, and designated citryl-CoA lyase (CCL). The citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i). citryl-CoA formation by citryl-CoA synthetase (CCS) and (ii). citryl-CoA cleavage by CCL. Purified CCL gave a single 30 kDa band in SDS-PAGE and gel filtration chromatography indicated that the native state of the enzyme exists as a trimer (alpha(3)). Citryl-CoA lyase showed low citrate synthase (CS) activity. Using an oligonucleotide probe, the corresponding gene was cloned and sequenced. The gene was expressed in Escherichia coli and recombinant CCL was also purified. The CCL protein sequence showed similarity to the C-terminal regions of ATP citrate lyase (ACL) and CS sequences in the database. By further sequence comparisons, the phylogenetic relationship between CCS, CCL, ACL and CS was investigated.