scholarly journals Conversion of 5-aminolaevulinate into haem by liver homogenates. Comparison of rat and chick embryo

1981 ◽  
Vol 198 (3) ◽  
pp. 595-604 ◽  
Author(s):  
J F Healey ◽  
H L Bonkowsky ◽  
P R Sinclair ◽  
J F Sinclair
Keyword(s):  
Chick Embryo ◽  
Rat Liver ◽  
Biosynthetic Pathway ◽  
Frozen Storage ◽  
Relevant Information ◽  
Porphobilinogen Deaminase ◽  
Embryo Liver ◽  
Liver Homogenates ◽  
Rate Limiting ◽  
Kinetics Of

1. We have studied the kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of livers of rats and chick embryos. Homogenates of fresh liver from both species efficiently convert 5-aminolaevulinate into haem. After frozen storage for 1 year, homogenates of rat, but not chick, liver have decreased rates of formation of haem with accumulation of more protoporphyrin. The rate of haem formation after storage is restored by addition of Fe2+ and menadione. 2. At all initial concentrations of 5-aminolaevulinate tested (2 microM-1 mM), homogenates of rat liver accumulate less protoporphyrin than haem. In contrast, homogenates of chick embryo liver accumulate more protoporphyrin than haem at concentration of 5-aminolaevulinate greater than 10 microM. Conversion of protoporphyrin into haem by homogenates of fresh or frozen chick embryo liver is not increased by addition of Fe2+. 3. Homogenates of liver from both species accumulate porphobilinogen; the kinetic parameters for this process reflect those of 5-aminolaevulinate dehydratase. 4. The results show that the rate-limiting enzyme for the hepatic conversion of 5-aminolaevulinate into protoporphyrin is porphobilinogen deaminase. In addition, chick liver, compared with rat liver, has only about one-fifth the activity of ferrochelatase, the final enzyme of the haem biosynthetic pathway, which inserts Fe2+ into protoporphyrin to form haem. 5. Comparison of these results with previous studies indicates that the homogenate system described here provides physiologically and clinically relevant information for study of hepatic haem synthesis and its control.

CHOLESTEROL BIOSYNTHESIS: THE STARVATION BLOCK

1957 ◽  
Vol 35 (1) ◽  
pp. 615-623
Author(s):  
J. F. Scaife ◽  
B. B. Migicovsky
Keyword(s):  
Experimental Evidence ◽  
Rat Liver ◽  
Biosynthetic Pathway ◽  
Glutaric Acid ◽  
Cholesterol Biosynthesis ◽  
Metabolic Block ◽  
Liver Homogenates ◽  
Acidic Compound

Partial localization of the metabolic block in cholesterol biosynthesis from acetate by starved rat liver homogenates has been achieved. Experimental evidence indicates that this block is located in the biosynthetic pathway between β-hydroxy-β-methyl glutaric acid and squalene. Fractionation and comparative chromatographic examination of incubated homogenates from starved and normal rats failed to reveal any accumulation of an appreciably radioactive intermediate as a result of the blocked biosynthetic pathway in the starved animal. A strongly labelled acidic compound has been isolated in minute amounts from incubated homogenates of both starved and normal rats. This is readily incorporated into cholesterol by liver homogenates from normal, but not from starved rats. Its identity has as yet not been established.

Kinetics of in vitro nitro reduction of 2,4-dinitrophenol by rat liver homogenates

1974 ◽  
Vol 27 (1) ◽  
pp. 140-144 ◽  
Author(s):  
Julie L. Eiseman ◽  
Perry J. Gehring ◽  
James E. Gibson
Keyword(s):  
Rat Liver ◽  
Liver Homogenates ◽  
Kinetics Of ◽  
Nitro Reduction

Porphyrinogenic effects in chick embryo liver cell culture of chloramphenicol analogues that are mechanism-based inactivators of cytochrome P-450

1991 ◽  
Vol 69 (4) ◽  
pp. 526-530 ◽  
Author(s):  
R. P. Green-Thompson ◽  
D. S. Riddick ◽  
J. E. Mackie ◽  
G. S. Marks ◽  
J. R. Halpert
Keyword(s):  
Cell Culture ◽  
Chick Embryo ◽  
Rat Liver ◽  
Liver Cell ◽  
Prosthetic Group ◽  
Uroporphyrinogen Decarboxylase ◽  
Compound A ◽  
Liver Cell Culture ◽  
Embryo Liver ◽  
Cytochrome P 450

Structural analogues of chloramphenicol (CAP) cause mechanism-based inactivation of rat liver cytochrome P-450 (P450) either via protein acylation or destruction of the heme prosthetic group. The goal of the present work was to determine whether CAP analogues that cause loss of the P450 heme moiety also cause porphyrin accumulation in chick embryo liver cell culture. The porphyrin profiles produced by exposure of cells to CAP analogues (160 μM) were determined by high-performance liquid chromatography with fluorescence detection. Of three CAP analogues that do not cause loss of the heme moiety of rat liver P450IIB1, two dichloroacetamides were not porphyrinogenic. The third compound, a chlorofluoroacetamide, caused porphyrin accumulation. This result may be due to the presence of P450 isozymes in chick embryo hepatocytes, distinct from rat liver P450IIB1, that are susceptible to destruction by this analogue. Of four CAP analogues that inactivate rat liver P450IIB1 with concomitant heme loss, a dichloroacetamide and two chlorofluoroacetamides caused porphyrin accumulation. The remaining compound, a monochloroacetamide, was not porphyrinogenic, perhaps because the P450 apoprotein cannot be reconstituted with fresh heme drawn from the regulatory "free heme pool" following inactivation by this analogue. Alternatively, there may be no P450 isozyme in chick embryo liver cell culture that is susceptible to inactivation by this compound.Key words: cytochrome P-450, chloramphenicol, chick embryo hepatocyte, mechanism-based inactivation, uroporphyrinogen decarboxylase.

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture

1989 ◽  
Vol 67 (3) ◽  
pp. 246-249 ◽  
Author(s):  
C. A. James ◽  
G. S. Marks
Keyword(s):  
Chick Embryo ◽  
Protein Concentration ◽  
Enzyme Assay ◽  
Uroporphyrinogen Decarboxylase ◽  
Optimum Ph ◽  
Embryo Liver ◽  
Liver Homogenates ◽  
Previous Demonstration ◽  
Sodium Phenobarbital ◽  
Cytochrome P 450

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10 000 g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein∙min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3′4,4′-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This result accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.Key words: uroporphyrinogen decarboxylase, enzyme inhibition, chick embryo hepatocytes, tetrachlorobiphenyl, porphyria.

CHOLESTEROL BIOSYNTHESIS: THE STARVATION BLOCK

1957 ◽  
Vol 35 (8) ◽  
pp. 615-623 ◽  
Author(s):  
J. F. Scaife ◽  
B. B. Migicovsky
Keyword(s):  
Experimental Evidence ◽  
Rat Liver ◽  
Biosynthetic Pathway ◽  
Glutaric Acid ◽  
Cholesterol Biosynthesis ◽  
Metabolic Block ◽  
Liver Homogenates ◽  
Acidic Compound

Partial localization of the metabolic block in cholesterol biosynthesis from acetate by starved rat liver homogenates has been achieved. Experimental evidence indicates that this block is located in the biosynthetic pathway between β-hydroxy-β-methyl glutaric acid and squalene. Fractionation and comparative chromatographic examination of incubated homogenates from starved and normal rats failed to reveal any accumulation of an appreciably radioactive intermediate as a result of the blocked biosynthetic pathway in the starved animal. A strongly labelled acidic compound has been isolated in minute amounts from incubated homogenates of both starved and normal rats. This is readily incorporated into cholesterol by liver homogenates from normal, but not from starved rats. Its identity has as yet not been established.

scholarly journals Conversion of 5-aminolaevulinate into haem by homogenates of human liver. Comparison with rat and chick-embryo liver homogenates

1985 ◽  
Vol 227 (3) ◽  
pp. 893-901 ◽  
Author(s):  
H L Bonkovsky ◽  
J F Healey ◽  
P R Sinclair ◽  
J F Sinclair
Keyword(s):  
Chick Embryo ◽  
Human Liver ◽  
Liver Homogenate ◽  
Iron Chelator ◽  
Experimental Models ◽  
Ph Optimum ◽  
Ph Values ◽  
Embryo Liver ◽  
Liver Homogenates ◽  
Human Livers

To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.

Patterns of porphyrin accumulation in response to chemicals in chick embryo liver cells

1983 ◽  
Vol 61 (6) ◽  
pp. 546-553 ◽  
Author(s):  
G. S. Marks ◽  
S. B. Follows ◽  
D. T. Zelt ◽  
S. P. C. Cole
Keyword(s):  
Chick Embryo ◽  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Liver Cells ◽  
Uroporphyrinogen Decarboxylase ◽  
Coproporphyrinogen Oxidase ◽  
Site Of Action ◽  
Endogenous Substrate ◽  
Embryo Liver ◽  
Ala Synthetase

Four patterns of porphyrin accumulation were observed by high-pressure liquid chromatography when chemicals were added to chick embryo liver cells. These patterns provide a guide to the site of action of the chemicals. Protoporphyrin accumulated in response to 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC), a result consistent with its ability to inhibit ferrochelatase. Uroporphyrin and heptacarboxylic acid porphyrin accumulated in response to 3,3′,4,4′-tetrachlorobiphenyl, 2,2′,4,4′,6,6′-hexachlorobiphenyl, and 3,5-diethoxycarhonyl-2,4,6-trimethylpyridine, a result suggesting inhibition of uroporphyrinogen decarboxylase. Coproporphyrin was the major porphyrin to accumulate in response to allylisopropylacetamide, aromatic amides, and steroids, a result suggesting inhibition of coproporphyrinogen oxidase. A mixture of uroporphyrin, heptacarboxylic acid porphyrin and coproporphyrin accumulated in response to aromatic di- and mono-esters, aliphatic diesters, and aliphatic amides. The pattern observed after addition of excess δ-aminolevulinic acid (ALA) the endogenous substrate of the pathway to the cells was proto- > copro- > uro-porphyrin. This pattern resembled that produced by DDC but by none of the other chemicals. It was concluded that porphyrin accumulation can not be attributed solely to the induction of ALA-synthetase. It appears that porphyrin-inducing chemicals exert an additional effect on one or other of the enzymes of the heme biosynthetic pathway.

VANILLINSÄURE ALS ENDPRODUKT DES STOFFWECHSELS VON ADRENALIN UND NORADRENALIN. II.

1963 ◽  
Vol 44 (1) ◽  
pp. 101-106 ◽  
Author(s):  
Wilhelm Dirscherl ◽  
Helmut Thomas
Keyword(s):  
Rat Liver ◽  
Paper Chromatography ◽  
Column Chromatography ◽  
Vanillic Acid ◽  
Hippuric Acid ◽  
Single Spot ◽  
Solvent Systems ◽  
Perfusion Experiment ◽  
Kinetics Of

ABSTRACT Perfusion of rat liver with vanillic acid yielded only one metabolite. In paper chromatography with three different solvent systems, the substance showed the same RF-values as vanillyolglycine (3-methoxy-4-hydroxyhippuric acid) and in mixed chromatograms there was only one single spot. After separation by column chromatography, the UV- and IRspectra of the reaction product were identical with those of 3-methoxy4-hydroxy-hippuric acid. During the perfusion experiment, the kinetics of the conjugation were investigated.

Kinetics of oestradiol-dependent induction of the prolactin receptor in rat liver

1988 ◽  
Vol 117 (4_Suppl) ◽  
pp. S88
Author(s):  
R. GÜNTHER ◽  
H. C. BLOSSEY
Keyword(s):  
Rat Liver ◽  
Prolactin Receptor ◽  
Kinetics Of
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