Cloning and nucleotide sequence of human liver cDNA encoding for cystathionine gamma-lyase.

We have cloned and sequenced a full-length cDNA (1083 bp) encoding the human liver cystathionine-gamma-lyase enzyme (cystathionase). The human cystathionase sequence presented a substantial deletion of 132 bases (44 amino acids) compared to that reported for rat cystathionase, and of 135 bases (45 amino acids) compared to that reported for yeast cystathionase. After re-alignment for the missing nucleotides, the human cDNA sequence shows significant amino acid homology to that for the rat enzyme (85%) and the yeast enzyme (50%). A search for an undeleted cDNA, by the polymerase chain reaction, yielded a second clone which contained the missing 132 bases. Flanking nucleotides in the latter clone were identical to those in the cDNA clone containing the deletion. The two forms of human cystathionase deduced from the two cDNA clones may be derived from two different genes or may be splice variants.     

MalY of Escherichia coli is an enzyme with the activity of a beta C-S lyase (cystathionase).

The Escherichia coli maltose system consists of a number of genes whose products are involved in the uptake and metabolism of maltose and maltodextrins. MalT is the central positive gene activator of the regulon and is, together with the cyclic AMP-catabolite gene activator protein system, necessary for the expression of the maltose genes. Expression of malY, a MalT-independent gene, leads to the repression of all MalT-dependent genes. We have purified MalY to homogeneity and found it to be a pyridoxal-5-phosphate-containing enzyme with the enzymatic activity of a beta C-S lyase (cystathionase). MalY is a monomeric protein of 42,000 to 44,000 Da. Strains expressing MalY constitutively abolish the methionine requirement of metC mutants. The enzymatic activity of MetC, the cleavage of cystathionine to homocysteine, ammonia, and pyruvate, can be catalyzed by MalY. However, the cystathionase activity is not required for the function of MalY in repressing the maltose system. By site-directed mutagenesis, we changed the conserved lysine residue at the pyridoxal phosphate binding site (position 233) of MalY to isoleucine. This abolished beta C-S lyase activity but not the ability of the protein to repress the maltose system. Also, the overexpression of plasmid-encoded metC did not affect mal gene expression, nor did the deduced amino acid sequence of MetC show homology to that of MalY.     

Induction of cystathionase in foetal rat liver explants effects of dexamethasone, N6,O2′-dibutyryladenosine 3′,5′-monophosphate and glucagon in vitro

At the 18th day of gestation and thereafter foetal rat liver explants in organ culture showed the competence to respond to dexamethasone by increased cystathionase activity, whereas the ability to respond to dibutyryl cyclic AMP or glucagon became evident at a later developmental stage (during the last 2 days prior to term). Simultaneous incubation with cycloheximide inhibited the stimulatory effect of these agents on foetal rat liver cystathionase activity in vitro. Dexamethasone and glucagon were both capable of increasing liver cystathionase activity both in newborn and 3-day-old animals in vivo.     

Studies on cystathionase activity in rat liver and brain during development. Effects of hormones and amino acids in vivo

High activity of cystathionase was present in rat liver but only low amounts of activity in rat brain during development. Triamcinolone had no effect on liver cystathionase activity in foetuses but increased the enzyme activity significantly in postnatal rats. l-Thyroxine decreased liver cystathionase activity significantly in newborn rats; administration of pyridoxal 5'-phosphate did not prevent this effect. l-Methionine significantly increased liver cystathionase activity in newborn rats.     

Cystathionase: high-performance liquid chromatography. Molecular cloning in lambda gt11. Nonradioactive immunodetection of fusion protein

A method of purification of rat liver cystathionase by high-performance liquid chromatography (HPLC) utilizing non-ideal gel filtration method is proposed. Resolution factors-flow rate, pH values, ionic strength of the mobile phase-were optimized. Antibodies to the enzyme were purified using an immunosorbent synthesized on the basis of epoxylated Toyopearl-65. Radioimmunoassay and immunoblotting demonstrated antibody monospecificity towards cystathionase. These monospecific antibodies were utilized for detecting enzyme amounts (up to 30 pg) using the avidin-biotin system. Rat cDNA expression library in phage lambda gt11 was screened. The cystathionase cDNA clone was isolated, and the structure of the insert was determined.     

Nonequivalent binding sites in cystathionase. Nanosecond and steady fluorescence studies.

The analogs P-pyridoxyl-L-alanine and P-pyridoxyl-L-homoserine bind to the apoprotein of the enzyme cystathionase and inhibit the reactivation of enzymatic activity after addition of pyridoxyl-5-P. The binding of the inhibitors was monitored by measuring the fluorescence emitted by the P-pyridoxyl moiety at 395 nm (excitation 325 nm). The fluorometric titration results indicate the presence of nonequivalent binding sites in the apoprotein. A model based on two classes of independent binding sites fits the fluorometric data reasonably well. The presence of nonequivalent fluorescent sites in reduced cystathionase was also detected by nanosecond spectroscopy. In contrast to the model compound P-pyridoxyl-epsilon-lysine (tau equals 2.6 ns), the P-pyridoxyl residues of cystathionase display multiexponential fluorescence decay. Two fluorescence lifetimes (tau2 equals 4.1 ns and tau2 equals 15 ns) fit the deconvoluted decay results obtained by pulse fluorimetry. It is proposed that the P-pyridoxyl chromophores of reduced cystathionase have different environments.     

On the nature of the activating enzyme of the inactive form of delta-aminolevulinate synthetase in Rhodopseudomonas spheroides.

The activating enzyme of the inactive form of Fraction I of delta-aminolevulinate (ALA) synthetase [EC 2.3.1.37] in Rhodopseudomonas (R.) spheroides was purified about 1,000-fold from an extract of R. spheroides cells grown anaerobically in the light. The purification of the activating enzyme was achieved by fractionating the 100,000 X g supernatant fraction of the crude extract with ammonium sulfate and acetone, followed by Sephadex G-200 chromatography, pyridoxamine phosphate-Sepharose 4B chromatography, and preparative gel electrophoresis. The final preparation of the activating enzyme still contained a minor contaminant (less than 20%) as judged by disc gel electrophoresis. The activating enzyme exhibited cystathionase [EC 4.4.1.1] activity throughout the purification. These two enzyme activities were not separated at all during any step of the purification. An apparently homogeneous preparation of cystathionase [EC 4.4.1.8] purified from rat liver also exhibited activating activity in the presence of L-cystine. It was concluded that the activating enzyme is a cystathionase.     

Cystathionase deficiency: the effect of cofactor on the stability of normal and abnormal enzyme from lymphoid cell lines.

Heating at 70 degrees C with and without added pyridoxal phosphate (PLP) had strikingly different effects on cystathionase protein from normal long-term lymphoid cell lines and on the enzymes from cells derived from patients with vitamin-B6-responsive cystathioninuria. PLP added to extracts of normal cells afforded complete protection against heat inactivation, whereas inactivation of the cystathionase protein in extracts obtained from two cystathioninuric lines was greater in the presence of PLP than in its absence.     

Cystathionine as a precursor of methionine in Escherichia coli and Aerobacter aerogenes

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and Stanley K. Shapiro. Cystathionine as a precursor of methionine in Escherichai coli and Aerobacter aerogenes. J. Bacteriol. 92:1331-1336. 1966.-Cystathionine has been shown to be a precursor of methionine biosynthesis in Escherichia coli and Aerobacter aerogenes. A double enzyme assay was developed to show the formation of homocysteine from cystathionine. The results obtained support the concept that cystathionine serves as a precursor of methionine via the intermediate formation of homocysteine. The latter compound is methylated by the homocysteine methyltransferase of these microorganisms. Sulfhydryl and keto acid assays were used to demonstrate cystathionase activity. Methionine represses both homocysteine methyltransferase formation and cystathionase formation. However, the presence of methionine in reaction mixtures resulted in product inhibition of homocysteine methyltransferase activity, but not of cystathionase activity.     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

Cat liver cystathionase.

Cat liver cystathionase was about 300-fold purified in comparison with the supernatant of the homogenate, and the characteristics were compared with those of rat. Optimum pHs for several substrates were found to be somewhat lower, and isoelectric point remarkably lower, in cat than in rat. Molecular weight of the cat liver cystathionase was about 158,000.     

Effect of methionine and vitamin B-12 on the activities of methionine biosynthetic enzymes in metJ mutants of Escherichia coli K12

The effects of supplementation of growth medium with high concentrations of methionine (5 mm) and/or vitamin B₁₂ (10 nm) on the activities of five enzymes of the methionine regulon were measured in wild-type Escherichia coli K12, a metJ prototrophic and three metJ methionine auxotrophic derivatives. Growth on vitamin B₁₂ causes lowering of the activities of the non-B₁₂ methyltransferase while growth on methionine causes elevation of its activity in all four metJ mutants. The previous observation that this enzyme is not repressed by vitamin B₁₂ addition in metH mutants together with our observation that vitamin B₁₂ causes repression in mutants (metF) unable to synthesize the donor for homocysteine methylation supports the model of Kung et al. (10) that the holo-B₁₂-methyltransferase functions as a repressor of synthesis of the non-B₁₂-methyltransferase. Growth on methionine causes lowering of cystathionase activity, and growth on vitamin B₁₂ results in elevation of cystathionase activity in a metJ prototroph and one metJ auxotroph. The metJmetA strain (RG326) has a higher than normal level of cystathionase while the metJmetF strain (RG191) has lower than normal cystathionase activity. These results indicate the existence of a metJ independent system that modulates the activity of cystathionase possibly in response to changes in concentration of unidentified metabolite(s).     

The novel product of a five-exon stargazin-related gene abolishes Ca(V)2.2 calcium channel expression

We have cloned and characterized a new member of the voltage-dependent Ca(2+) channel gamma subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential gamma subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated gamma(7). Expression of human gamma(7) has been localized specifically to brain. N-type current through Ca(V)2.2 channels was almost abolished when co-expressed transiently with gamma(7) in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that gamma(7) has this effect by causing a large reduction in expression of Ca(V)2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed gamma(7) was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like gamma subunits, different gene structure and the unique functional properties of gamma(7) imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.     

Persulfide generated from L-cysteine inactivates tyrosine aminotransferase. Requirement for a protein with cysteine oxidase activity and gamma-cystathionase

Liver cytosols contain factors that produce an inhibitor of tyrosine aminotransferase and other enzymes when incubated with L-cysteine or L-cystine. Cystine-dependent inactivation was caused by cystathionase and required pyridoxal 5'-phosphate, but a second protein was needed to reconstitute cysteine-dependent inactivation. A cytosolic protein was isolated that oxidized free cysteine and brought about inactivation of tyrosine aminotransferase when coincubated with cystathionase. Hematin also oxidized cysteine, which led to cysteine-dependent inactivation of tyrosine aminotransferase in the presence of cystathionase. The inactivation of tyrosine aminotransferase involved three steps: initial oxidation of cysteine to form cystine; desulfuration of cystine catalyzed by cystathionase to form the persulfide, thiocysteine; and reaction of thiocysteine (or products of its decomposition) with proteins to form protein-bound sulfane. Since dithiothreitol reactivated tyrosine aminotransferase, the sulfane probably inactivated the enzyme by oxidation of thiol groups. The present results do not indicate whether the cysteine oxidase activity is enzymatic nor do they prove which form of polysulfide inactivates tyrosine aminotransferase. Reduced glutathione greatly slowed the rates at which sulfane accumulated and at which tyrosine aminotransferase was inactivated. Incubation of DL-cystathionine with liver cytosols led to formation of cysteine, which was oxidized and cleaved to form persulfide, and caused inactivation of tyrosine aminotransferase. Thus, sulfane sulfur that is generated by an enzyme of the transulfuration pathway inactivates a transaminase by nonselective oxidation of enzyme-bound thiol groups.     

Effects of thiazolidine-4(R)-carboxylates and other low-molecular-weight sulfur compounds on the activity of mercaptopyruvate sulfurtransferase, rhodanese and cystathionase in Ehrlich ascites tumor cells and tumor-bearing mouse liver

Summary Changes of the specific activity of 3-mercaptopyruvate sulfurtransferase (MPST), rhodanese and cystathionase in Ehrlich ascites tumor cells (EATC) and tumor-bearing mouse liver after intraperitoneal administration of thiazolidine derivatives, L-cysteine, D,L-methionine, thiocystine or thiosulfate were estimated. Thiazolidine derivatives used were: thiazolidine-4-carboxylic acid (CF), 2-methyl-thiazolidine-2,4-dicarboxylic acid (CP) and 2-methyl-thiazolidine-4-carboxylic acid (CA). In the liver, the activity of MPST was significantly increased by all the studied compounds, whereas the activity of rhodanese was by CF and thiocystine and that of cystathionase was by the administration of cysteine and CP. Un the other hand, cysteine lowered the rhodanese activity and the activity of cystathionase was decreased by the administration of methionine and thiocystine. Activities of MPST and rhodanese were even lower in EATC than those in the liver of tumor-bearing mouse and the activity of cystathionase in EATC was not be detected. The thiazolidine derivatives significantly increased the level of MPST activity in EATC, but decreased the rhodanese activity. Thiosulfate also increased the activity of MPST to a lesser degree, but cysteine, methionine and thiocystine gave little change in the activity. The rhodanese activity in EATC was slightly increased only by thiocystine. These findings suggest that the sulfur metabolism in the tumor-bearing mouse liver is different from that in the normal mouse liver, and that sulfur compounds are minimally metabolized to sulfane sulfur, a labile sulfur, in EATC.     

Activities of Cysteine Synthetase and Cystathionase in Bacilhis subtilis during Sporulation

Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A–50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.

The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores.     

Effects of inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat.

The effects of alterations in sulfur metabolism on hepatic and renal metallothionein and glutathione metabolism were studied in the adult rat using inhibition of two enzymes of these pathways, hepatic cystathionase and renal gamma-glutamyl transpeptidase. Rats were fed a diet containing both methionine (0.66%) and cystine (0.20%) for 1 week before receiving three consecutive daily intraperitoneal injections of propargylglycine, a selective cystathionase inhibitor, at various doses (2.5-375 mumol/kg). When hepatic cystathionase was inhibited greater than 90% (greater than or equal to 50 mumol propargylglycine/kg), renal and hepatic metallothionein and hepatic glutathione were unaltered except at the highest dose. On the other hand, renal glutathione was increased two-fold with a concomitant decrease in renal gamma-glutamyl transpeptidase activity (50% of control). In another experiment, when renal gamma-glutamyl transpeptidase was inhibited greater than 90% with three consecutive daily injections of acivicin, a selective gamma-glutamyl transpeptidase inhibitor (10 mg/kg IP), renal glutathione content was unaltered while hepatic glutathione was decreased. Renal and hepatic metallothionein were not changed. Thus, the cysteine pools for metallothionein and glutathione appear unrelated under the present experimental conditions. In addition, following either proparglyglycine or acivicin injections, renal and hepatic glutathione pools appear to be altered differently. These results suggest that renal glutathione may be preferentially maintained even when hepatic glutathione is decreased.     

Accumulation of cystine auxotrophic thymocytes accompanying type C viral leukemogenesis in the mouse.

Certain continuous lymphoid and myeloid tumor cell lines of rodent origin are unable to grow in tissue culture in the absence of pre-formed L-cystine (CYS). In contrast, three NZB murine lymphoid cell lines obtained from NZB mice free of hematopoietic neoplasm can grow as well in cystine-deficient media containing L-cystathionine (CSN), the immediate precursor of CYS in the biosynthetic pathway, as in cystine sufficient medium. The former class of cells is, therefore, CYS auxotrophs (CYS-) and the latter CYS prototrophs (CYS+). Compared to CYS+ cells, the CYS- lines appear to be relatively deficient in the enzyme cystathionase, which catalyzes the cleavage of CSN to CYS and alpha-ketobutyrate. Using protein synthetic capacity as a criterion, normal thymocytes from mixed-bred Swiss mice behave like CYS prototrophs, while those from littermates bearing Moloney type C virus-induced thymic tumors behave like CYS auxotrophs. The former are also characterized by substantially higher levels of cystathionase than the latter. Extracts of thymocytes from tumor-free AKR mouse thymus are also characterized by higher levels of cystathionase activity than extracts of spontaneous AKR thymomas. Exogenous in vitro type C virus infection of a CYS+ cell results in vigorous virus production but no concomitant reduction in cystathionase activity. Thus viral replication alone in any random lymphoid cell is not sufficient to alter the enzyme level. The data therefore suggests that CYS auxotrophy may closely accompany neoplastic transformation of certain hematopoietic cells in vivo, including that induced by certain "thymic" type C viruses.     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.