Dehydrase Activity Research Articles

Effect of methylcobalamin-C-10-Br from stinky tofu on inhibition of Listeria monocytogenes and alteration of microbiota in an in vitro colonic simulation

In this study, it was found that methylcobalamin-C-10-Br (MeCbl-C-10-Br) from stinky tofu exerts an inhibitory effect on Listeria monocytogenes, and the minimum inhibitory concentration was 0.125 ng/mL. Here, we studied the effect of MeCbl-C-10-Br on the adenosylcobalamin (AdoCbl)-dependent enzyme glycerol dehydratase, the methylcobalamin (MeCbl)-dependent enzyme methionine synthetase and three types of vitamin B12 riboswitches from L. monocytogenes. The results show that MeCbl-C-10-Br inhibits about 100% of glycerin dehydrase activity and about 37% methionine synthase activity. While the expression of the downstream genes of the vitamin B12 riboswitch is suppressed, the ratios of inhibition of transport, synthetic, and functional vitamin B12 riboswitches from 1.00 μg/mL MeCbl-C-10-Br are approximately 60%, 30%, and 40%, respectively, which suggests that MeCbl-C-10-Br effectively controls the growth of L. monocytogenes. According to the results of the simulated fermentation, MeCbl-C-10-Br did not destroy the balance of intestinal microbes after ingestion, which indicates that MeCbl-C-10-Br is a safe antibacterial agent and can be applied to food in the future.

Catalytic mechanism of serine racemase from Dictyostelium discoideum

The eukaryotic serine racemase from Dictyostelium discoideum is a fold-type II pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes racemization and dehydration of both isomers of serine. In the present study, the catalytic mechanism and role of the active site residues of the enzyme were examined by site-directed mutagenesis. Mutation of the PLP-binding lysine (K56) to alanine abolished both serine racemase and dehydrase activities. Incubation of D- and L-serine with the resultant mutant enzyme, K56A, resulted in the accumulation of PLP-serine external aldimine, while less amounts of pyruvate, α-aminoacrylate, antipodal serine and quinonoid intermediate were formed. An alanine mutation of Ser81 (S81) located on the opposite side of K56 against the PLP plane converted the enzyme from serine racemase to L-serine dehydrase; S81A showed no racemase activity and had significantly reduced D-serine dehydrase activity, but it completely retained its L-serine dehydrase activity. Water molecule(s) at the active site of the S81A mutant enzyme probably drove D-serine dehydration by abstracting the α-hydrogen in D-serine. Our data suggest that the abstraction and addition of α-hydrogen to L- and D-serine are conducted by K56 and S81 at the si- and re-sides, respectively, of PLP.

Inhibition Effect of Poly(γ-glutamic acid) on Lead-Induced Toxicity in Mice

The objectives of this study were to evaluate the efficiency in treatment of lead-induced intoxication in mice with γ-PGA as chelating agent and compare with the drug (meso-2,3-dimercaptosuccinic acid). The results showed the incorporation of γ-PGA at 200 and 400 mg/kg could reduce the accumulation of lead in the liver, heart, and testis; however, the latter was more effective in decreasing the lead content in the kidney and spleen. Nevertheless, both doses failed to inhibit the lead accumulation in the lung and brain. Additionally, both doses of γ-PGA could reduce TBARs in the kidney and brain, as well as elevate δ-aminolevulinic acid dehydrase (δ-ALAD) activity in blood and decrease glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH) activities in the serum. For hematological parameters, both white blood cells (WBCs) and hematocrite (HCT) were raised by 400 mg/kg of γ-PGA, while for both doses of γ-PGA, a slight decline in hemoglobin (HGB), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) was observed, with the red blood cells (RBCs) being unaffected.

A Retro-Evolution Study of CDP-6-deoxy-d-glycero-l-threo-4-hexulose-3-dehydrase (E1) from Yersinia pseudotuberculosis: Implications for C-3 Deoxygenation in the Biosynthesis of 3,6-Dideoxyhexoses

CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which catalyzes C-3 deoxygenation of CDP-4-keto-6-deoxyglucose in the biosynthesis of 3,6-dideoxyhexoses, shares a modest sequence identity with other B6-dependent enzymes, albeit with two important distinctions. It is a rare example of a B6-dependent enzyme that harbors a [2Fe-2S] cluster, and a highly conserved lysine that serves as an anchor for PLP in most B6-dependent enzymes is replaced by histidine at position 220 in E1. Since alteration of His220 to a lysine residue may produce a putative progenitor of E1, the H220K mutant was constructed and tested for the ability to process the predicted substrate, CDP-4-amino-4,6-dideoxyglucose, using PLP as the coenzyme. Our data showed that H220K-E1 has no dehydrase activity, but can act as a PLP-dependent transaminase. However, the reaction is not catalytic since PLP cannot be regenerated during turnover. Reported herein are the results of this investigation and the implications for the role of His220 in the catalytic mechanism of E1.

철분과 운동이 흰쥐의 헴 생합성과 지질 대사에 미치는 영향

본 연구는 식이 철과 운동이 흰쥐의 헴 생합성과 지질대사에 미치는 영향을 살펴보고자 수행되었다. 쥐는 식이 철의 수준에 따라 철결핍군(Fe^-, 5 ppm)과 철 충분군(Fe^+, 50 ppm)으로 나누고 규칙적인 운동 여부에 따라 운동군과 대조군으로 나누었다. 실험 식이의 공급과 운동을 시킨 기간은 8주였으며, 운동군은 경사 6도에서 28 m/min의 속도로 1회 30분씩 일주일에 5회씩 트레이드밀 운동을 시켰다. 그 결과 흰쥐의 헴 생합성 정도는 운동을 한 군에서 유의적으로 증가하는 것으로 나타났다. 운동은 또한 체중 증가량을 낮추고, 부고환지방량을 감소시켰으며, 혈중 중성지방과 콜레스테롤 수치를 낮추고 HDL-콜레스테롤의 수치를 증가시켰다. 한편, 식이 철의 부족은 헤마토크리트와 헤모글로빈 농도를 감소시켰으며, 간 콜레스테롤의 축적을 증가시켰다. 앞으로 인체를 대상으로 철분 수준이 다른 상태에서의 운동이 헴의 생합성, 지질대사 및 젖산 농도 등에 어떠한 영향을 미치는지에 대한 연구가 더 많이 이루어져야 할 것으로 사료된다.

Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO(3) (-) dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 micromol H(+) (mg Chl)(-1) h(-1)) were observed in the presence of detergents (Triton X-100 or n-octyl-beta-D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 micromol H(+) (mg Chl)(-1) h(-1) which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rumen bacterium Lachnospira multiparus.

Lachnospira multiparus belongs to the main rumen pectinolytic bacteria. Its carbohydrate metabolism was studied in growth experiments on laboratory fermenters, and using assays of activities of enzymes involved in pectin fermentation. The type strain of this species and two substrates were used. Lachnospira multiparus ATCC 19207 grew on pectin and glucose at a similar rate and had no preference for one or the other substrate. Pectin-grown cultures, however, produced significantly more acetate and less formate, lactate, ethanol, hydrogen, cell dry matter and protein than corresponding cultures grown on glucose. Extracellular exopectate lyase (EC 4.2.2.9) was the principal enzyme degrading the pectin macromolecule. Cell extracts possessed 2-keto-3- deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) and fructosediphosphate aldolase (EC 4.1.2.13) activity. The former enzyme catalyses the final reaction in the Entner-Doudoroff pathway; the latter is the key enzyme of glycolysis and the pentose phosphate pathway. These results are consistent with the assumption that acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway. Phosphogluconate was not metabolized by cell extracts of the strain studied. This suggests that the conventional Entner-Doudoroff pathway of glucose utilization does not operate in this bacterium, presumably because of the lack of 6-phosphogluconate dehydrase (EC 4.2.1.12) activity.

Fats and Fatty Acids as Growth Factors for Lactobacillus delbrueckii

The effects of various fats and fatty acids on the growth of Lactobacillus delbrueckii strains have been studied using modified MRS broth without Tween 80 as a basic growth medium. Among the six L. delbrueckii strains studied all except one strain required Tween 80 or Tween 20 as a fatty acid supplement for the growth. Tween 40 and Tween 60, which contain solely medium and long chain saturated fatty acids, inhibited the growth of all L. delbrueckii strains when present as a sole fat supplement in MRS broth. Free oleic acid but not free lauric acid could substitute Tween 80 or Tween 20 supplement suggesting that unsaturated fatty acids are essential growth factors for most L. delbrueckii strains. Among the natural food oils tested, the oils containing the lowest amounts of saturated long chain fatty acids promoted the growth of L. delbrueckii most effectively. Especially cellular C18:1 and C19 cyclopropane fatty acid contents of L. delbrueckii were strongly affected by exogenous fatty acid composition and by strain suggesting genetic diversity and polymorphism among the genes encoding and/or regulating cyclopropane synthase. In addition obviously most if not all L. delbrueckii strains lack particular synthase, desaturase and/or dehydrase activities required for de novo synthesis of long chain unsaturated fatty acids. These biochemical features could be used as informative chemotaxonomic characteristics for L. delbrueckii starter strain identification and selection.

Molecular Cloning, Sequencing, and Expression of the Genes Encoding Adenosylcobalamin-dependent Diol Dehydrase of Klebsiella oxytoca

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.

An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis

Escherichia coli strain SM101 harbors a temperature-sensitive allele (lpxA2) of the gene encoding UDP-Glc-NAc acyltransferase (the first enzyme of the lipid A pathway). SM101 is temperature-sensitive for lipid A biosynthesis and growth. To determine whether or not E. coli mutants lacking lipid A can be isolated, we examined temperature-resistant revertants of SM101. All regained the ability to synthesize lipid A. However, some were not true revertants but had acquired mutations in a neighboring gene (orf17), while retaining the original lpxA2 lesion. Cell extracts of such revertants displayed 2-5 fold reductions in the specific activity of (3R)-hydroxymyristoyl-ACP dehydrase. Wild-type cells that overproduced the protein encoded by orf17 overproduced (3R)-hydroxymyristoyl-ACP dehydrase activity as much as 170-fold, suggesting that orf17 is the structural gene for the dehydrase. The proposed function of orf17 is further supported by its sequence similarity to fabA, the structural gene for (3R)-hydroxydecanoyl dehydrase of E. coli. We suggest that bypass of the lpxA2 phenotype by mutations in orf17 may be due to an increased (3R)-hydroxymyristoyl-ACP pool. The orf17 gene (which we now designate fabZ) is not regulated by fadR. However, orf17 may be related to sefA, a suppressor of certain lesions in the cell division/lipid A biosynthesis gene, envA.

Effect of environmental exposures to lead and cadmium on human lymphocytic detoxifying enzymes

Lead (Pb) is among the most toxic heavy elements in the atmosphere. Aerosol lead enters the human blood stream by way of the respiratory tract and indirectly, by surface disposition in the alimentary tract followed by adsorption. Lead pollution is also known to occur through its presence in petrol, pain, glazed vessels and solder. Atmospheric lead pollution may be predominantly high around factories manufacturing Pb alloys. Lead toxicity is associated with inhibition of [alpha]-aminolevulinic acid dehydrase (ALAD) activity, rise in the blood porphyrin, inhibition of ATPase in erthrocytes, decreased blood haemoglobin and anemia. Elevated lead concentrations in pregnant women have been shown to cause hypertension and birth defects. Lead is also known to interact with other elements such as Fe, Zn, Ca and Cu in biological systems. Cadmium (Cd) is not essential for human body. It enters the human environment as a contaminant. Human intake of Cd is chiefly through the food chain (about 400-500 [mu]g/wk). Analysis of neuropsy material shows that smokers accumulate much more Cd than nonsmokers. Chronic Cd poisoning produces proteinuere and affects the proximal tubules of kidney, causing the formation of kidney stones. The reported hypertensive effect of Cd in man has been associated with highmore » Cd/Zn ratio in kidney. Studies on air pollution have shown that Cd concentration in air could be positively correlated with heart disease, hypertension and arteriosclerosis. The present investigation was aimed at assessing the usefulness of human lymphocytic detoxicating enzyme activities and their ratios in an assessment of human health-risks during environmental exposures to Pb and Cd. The human subjects investigated comprised those exposed to highly contaminated lead and cadmium areas in the state of Maharashtra, India. 17 refs., 2 figs.« less

Heme biosynthesis in uremic patients on CAPD or hemodialysis

We have analyzed some parameters of porphyrin metabolism in 60 patients with end-stage renal failure, 20 of them on CAPD and the remaining on HD. In comparison with 56 control subjects, both groups of patients showed the three following findings: low erythrocyte aminolevulinate dehydrase activity, inhibition ability for the activity of this enzyme when their plasma was incubated in vitro with normal erythrocytes, and increased plasma porphyrin levels. Like anemia, these abnormalities were more remarkable in patients on HD who also exhibited increased erythrocyte protoporphyrin levels and compensatory porphobilinogen deaminase activities. Mean weekly porphyrin removal through dialysate was higher in CAPD (90.8 micrograms) than in HD patients (30.4 micrograms). Dialysate and plasma porphyrins were correlated in both circumstances (r = 0.714, P < 0.01 and r = 0.637, P < 0.05, respectively). The less pronounced porphyrin abnormalities found in CAPD patients with respect to HD patients may be due to the more efficient capability of peritoneal dialysis for removing from plasma protein-bound substances, as porphyrins and inhibitors of aminolevulinate dehydrase or other enzymes involved in erythropoiesis. Since no close relationship was found between these abnormalities of porphyrin metabolism and hematocrit values, the anemia of uremia cannot be merely considered as a direct consequence of altered heme biosynthetic pathway.

Construction, expression, and characterization of a mutated animal fatty acid synthase deficient in the dehydrase function.

The location of the dehydrase domain in the multifunctional animal fatty acid synthase has been determined by engineering a fatty acid synthase mutant deficient in dehydrase activity. A full-length fatty acid synthase cDNA encoding a mutated histidine residue (His878-->Ala) was constructed and expressed in insect Sf9 cells using a baculoviral vector. The mutated recombinant fatty acid synthase retained all partial activities of the multifunctional complex except the dehydrase and was unable to synthesize fatty acids. beta-Hydroxybutyryl moieties were formed by the mutant fatty acid synthase from acetyl-CoA, malonyl-CoA, and NADPH and slowly released as the CoA thioester, confirming that this protein cannot perform the dehydration reaction. This finding points to an important catalytic role for His878 in the dehydration reaction and establishes that the dehydrase domain is located immediately adjacent to the carboxyl terminus of the transferase domain. Examination of the completed domain map for the animal fatty acid synthase indicates that the catalytic domains are clustered in two groups separated by a central structural core: the ketoacyl synthase, malonyl/acetyltransferase, and dehydrase in the amino-terminal half and the enoyl reductase, ketoreductase, acyl carrier protein, and thioesterase in the carboxyl-terminal half. A model is proposed in which the two centers for acyl chain initiation, elongation and termination, are formed by the cooperation of the three amino-terminal domains of one subunit with the four carboxyl-terminal domains of the other subunit.

The prevalence and retention of lead pellets in Japanese quail

Thirty-six Japanese quail (18 control birds, 18 lead-dosed birds) were used. The 18 quail were dosed with #4 lead weight that were orally inserted into the proventriculus. Delta-aminolevulunic acid dehydrase (ALAD) activity in erythrocytes in the dosed quail decreased 90% (p < 0.01) after one week as compared with the undosed quail. This inhibition of ALAD activity in erythrocytes indicates lead exposure. Radiographics were obtained at 0, 1, 4, 9, 22, and 32 days. The lead pellets remained in the gizzard and became smaller in 4 days. At day 22, after the lead treatment, in 8 quail of the treated quail 12, the lead pellets disappeared. At day 32, all lead pellets disappeared. These findings indicate that the ingested lead pellets are absorbed gradually in the intestine. The lead concentrations in the blood, liver, kidney, and femur of the lead-dosed quail were significantly higher than in the undosed quail until the 6th week. At week 2, the lead concentration of the proventriculus, gizzard, gizzard contents, duodenum, small intestine, and cecum in the dosed quail was significantly higher. Lead concentration of feces was significantly higher at weeks 2 and 4 (p < 0.01). Throughout this study, no lead pellets were found in the feces.

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.

A spectrophotometric assay for hydroperoxide lyase

AbstractSpectrophotometric assays for hydroperoxide lyase have traditionally measured the loss in absorption at 234 nm due to the disruption of conjugated diene in the fatty acid hydroperoxide. However, that assay does not distinguish between hydroperoxide lyase and hydroperoxide dehydrase activities, both of which cause a loss of conjugation in the substrate hydroperoxide. A new assay has been developed which is specific for hydroperoxide lyase. It is based on the ability of hydroperoxide lyase products, aldehydes and ω‐oxoacids, to serve as substrates for yeast alcohol dehydrogenase. Thus, the new hydroperoxide lyase assay is a coupled enzyme assay, which is conducted spectrophotometrically at 340 nm and measures the oxidation of nicotinamide adenenine dinucleotide, reduced form (NADH). In addition to its specificity for hydroperoxide lyase, the coupled assay allows higher concentrations of both fatty acid hydroperoxide substrate and crude enzyme extracts than the assay conducted at 234 nm.

Topography of Rat Hepatic Microsomal Enzymatic Components of the Fatty Acid Chain Elongation System

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.

Pathways of Fatty Acid Hydroperoxide Metabolism in Spinach Leaf Chloroplasts

The metabolism of 13-hydroperoxylinolenic acid was examined in protoplasts and homogenates prepared from mature leaves of spinach (Spinacia oleracea L.). Chloroplast membranes were the principal site for metabolism of the compound by at least two highly hydrophobic enzyme systems, hydroperoxide lyase and hydroperoxide dehydrase, the new name for an enzyme system formerly known as hydroperoxide isomerase and hydroperoxide cyclase. Hydroperoxide lyase was most active above pH 7 and could be separated from hydroperoxide dehydrase by anion exchange chromatography. Hydroperoxide dehydrase, measured by the formation of both alpha-ketol product and 12-oxo-phytodienoic acid, had its optimum activity in the range of pH 5 to 7. Lyase was more active than dehydrase activity when the enzymes were extracted by homogenization. The reverse was true when the enzyme activities were measured in protoplasts, which are isolated by gentle extraction methods. The variation in enzyme activity ratios with extraction methods suggests that hydroperoxide lyase is activated by plant injury and thus may function in a wound response. In the absence of injury, the normal pathway of fatty acid hydroperoxide metabolism is probably by hydroperoxide dehydrase activity. The molecular weights of both the lyase and dehydrase were approximately 220,000, as estimated by gel filtration.

Hepatic heme synthesis in a new model of experimental hemochromatosis: studies in rats fed finely divided elemental iron.

Rats fed chow containing finely divided elemental iron (from carbonyl-iron) develop hepatic iron overload resembling human hereditary hemochromatosis in that deposition of iron is primarily in periportal hepatocytes and with hepatic iron concentrations sufficiently high to be associated in the human disease with hepatic fibrosis or cirrhosis. In recent studies using this model, we reported changes in hepatic hemoproteins and heme oxygenase, the rate-controlling enzyme of heme breakdown. We now report effects of iron-loading on three enzymes of heme synthesis: 5-aminolevulinate synthase; the first and rate-controlling enzyme of the pathway, 5-aminolevulinate dehydrase (or porphobilinogen synthase), and uroporphyrinogen decarboxylase, the activity of which is decreased in porphyria cutanea tarda, a liver disease in which iron is known to play an important but still poorly understood role. Of the three enzymes, only activity of the dehydrase was altered by iron-loading: it was decreased significantly as early as 1 week after starting iron feeding, and with marked iron overload was 30 to 32% of control values. The degree of decrease was inversely related (r = -0.77 to -0.88) to the degree of iron overload and was partially reversed within 1 to 3 days when feeding of the iron-supplemented diet was stopped. The decrease in dehydrase activity was not attributable to lack of reduced glutathione or other disulfide-reducing agents or to zinc deficiency; nor was evidence found for inhibition by iron compounds or other possible inhibitors present in iron-loaded livers.(ABSTRACT TRUNCATED AT 250 WORDS)