Decrease of rat liver cysteine dioxygenase (cysteine oxidase) activity mediated by glucagon.

The hepatic cysteine dioxygenase activity of rats was markedly decreased by the intraperitoneal administration of glucagon. The enzyme activity was also decreased by either dibutyryl cyclic AMP or theophylline. The prior administration of actinomycin D completely blocked the glucagon-mediated decrease of enzyme activity, while administrations of this inhibitor of protein synthesis after glucagon injection did not block the decrease of enzyme activity. A single administration of actinomycin D resulted in a slight increase of cysteine dioxygenase activity in the rat liver. On the other hand, the injection of cycloheximide resulted in a rapid decrease of the hepatic cysteine dioxygenase with a half-life of 2.5 h. The half-life of the enzyme in rat liver after glucagon administration was one hour. The administration of hydrocortisone or insulin had no effect on the glucagon-mediated decrease of cysteine dioxygenase of rat liver. The enzyme activity of alloxan diabetic rat liver was almost the same as that of the intact rat liver. The evidence obtained here suggests that enhancement of degradation or inactivation of cysteine dioxygenase is responsible for the glucagon-mediated decrease of the enzyme activity in rat liver.     

Effects of several unusual sulfur-containing amino acids on rat liver cystathionine-gamma-lyase.

1. The mode of inhibition of rat liver cystathionine-gamma-lyase [L-cystathionine cysteine-lyase (deaminating), EC 4.4.1.1] was studied by using several unusual sulfur-containing amino acids newly found in this laboratory. Some cysteine conjugates (CMC, Beta-CEC, HCETC and HCPC) inhibited noncompetitively both homoserine dehydratase and diaminopropionate ammonia-lyase activities, and competitively gamma-cystathionase activity. CMTC exhibited a mixed type inhibition on both homoserine dehydratase and gamma-cystathionase activities, and a noncompetitive inhibition on the diaminopropionate ammonia-lyase activity. Some homocysteine conjugates (CMHC, beta-CEHC and HCEHC) inhibited competitively both the activity of homoserine dehydratase and of gamma-cystathionase, and exhibited a mixed type inhibition on the diaminopropionate ammonia-lyase activity. beta-CEC, CMHC and beta-CEHC were also effective inhibitors to cysteine desulfhydrase activity. 2. Among the other amino acids tested, DL-homocysteine and D-cysteine, irrespective of their concentration, exhibited a mixed type inhibition on the homoserine dehydratase activity. However, they promoted gamma-cystathionase activity at their lower concentrations and inhibited at their higher concentrations, more so than cystathionine. DL-alpha-Aminobutyric acid was a weak competitive inhibitor of the homoserine dehydratase, gamma-cystathionase and diaminopropionate ammonia-lyase activities. DL-alpha-Aminopimeric acid has the same chain length as beta-CEC, CMHC and CMTC, but it showed a very weak inhibitory effect compared with the latter sulfur-containing compounds. L-Methionine, DL-methionine sulfoxide, L-ethionine, L-cysteic acid, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, D-alanine, beta-alanine, L-ornithine and L-lysine had little or no effect on any activities of the enzyme preparation. These results were discussed in relation to the catalytic center of cystathionine-gamma-lyase.     

Down-regulation of rat liver beta-adrenergic receptors by cysteine

Down-regulation of hepatic beta-adrenergic receptors was indicated by a 56% decrease in the specific activity of 125I-iodocyanopindolol bound to rat liver membrane preparations from rats fed diets containing 15% of casein supplemented with cysteine, instead of methionine or unsupplemented. Down-regulation of hepatic beta-adrenergic receptors by cysteine appears to be mediated through an effect of cysteine on the tissue concentration of S-adenosyl methionine (SAM). The liver tissue concentration of SAM in rats fed cysteine-supplemented diets decreased 53% compared to those fed diets supplemented with methionine. The decrease in liver SAM in rats fed the diet supplemented with cysteine appears to reflect a non-competitive inhibition of methionine adenosyl-transferase by cysteine. Lineweaver-Burk plots demonstrated a dose-related Vmax response to cysteine but did not change the apparent Km at any concentration tested.     

ATP-stimulated uptake of S-(2,4-dinitrophenyl)glutathione by plasma membrane vesicles from rat liver

ATP-stimulated uptake of S-(2,4-dinitrophenyl)glutathione with a high activity of 0.35 nmol/min per mg protein is found in a rat liver plasma membrane vesicle preparation enriched in sinusoidal marker enzymes. Transport takes place into an osmotically active space. Vanadate and S-(azidophenacyl)glutathione inhibit transport, whereas Ca2+, EGTA and ouabain are without effect.     

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase).

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.     

Segments of paramyosin formed by cleavage at sites of cysteine residues.

The helical muscle protein beta-paramyosin of 200,000 was treated by the general method of G. R. Jacobson et al. (1973), J. Biol. Chem. 248, 6583) for cleavage of the polypeptide chain at the site of Cys residues. The protein cleaved into two segments: CCF-1 of 140,000 daltons and CCF-2 of 60,000 daltons. The two segments were separated and some properties were compared. Circular dichroism measurements indicated that CCF-1 was completely helical and that CCF-2 was 85% in the alpha-helical form. The molecular size, resistance to pepsin digestion, stability to heat and urea, and solubility of CCF-1 were all similar to corresponding properties of a pepsin-resistant segment PPC-1 described earlier (Cowgill, R. W. (1972), Biochemistry 11, 4532). By contrast, the properties of CCF-2 were distinctly different. It was concluded that the CCF-1 segment, like the PPC-1 segment, arose from the N-terminal two-thirds of the paramyosin molecule. The CCF-2 segment from the C-terminal one-third of paramyosin had limited solubility at neutral pH that matched the low solubility of paramyosin. It was concluded that the CCF-2 region is responsible for the self-aggregating tendency of paramyosin at neutral pH and low ionic strength.     

Two components of cysteine oxidase in rat liver

Purified cysteine oxidase in rat liver is composed of two distinct proteins. These proteins are able to be fractionated by DEAE-cellulose column chromatography. It appears that one of them is a catalytic protein named protein-B having tightly bound iron as a prosthetic group, while the other is either a modifier or activating protein named protein-A. Protein-B is found to exist in both an active and an inactive form. Inactive protein-B is activated by incubation with substrate cysteine under anaerobic condition. Activated protein-B alone exhibited an extremely low catalytic activity but in the presence of protein-A remarkable increase in activity was observed.     

Modification of polynucleotides by a fragment produced by enzymatic cleavage of S-(1, 2-dichlorovinyl)-L-cysteine

Polyribo- and polydeoxyribonucleotides were allowed to react with ³⁵S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in presence of a bovine kidney lyase yielding products which were substituted to varying degrees with an alkylating thiovinyl fragment (AF) released from DCVC. Polydeoxyribonucleotides were more extensively substituted than polyribonucleotides. Double stranded homopolymer pairs were much less effective as acceptors of (AF) than single stranded polymers. Nucleotide substitution occurred only at the polymer level. Enzymatic hydrolysis of (AF)-substituted polymers yielded dinucleotides which contained an (AF) fragment apparently covalently linked in unknown fashion. (AF)-substituted polynucleotides had reduced ability to form helical complexes with complementary polynucleotides, as revealed by hypochromicity, melting transition and renaturation.     

Specific enzymic cleavage of polypeptides at cysteine residues.

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the beta chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified cysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide.