Measurement of ALA synthase activity.

In most cells, δ-aminolevulinate (ALA) synthase is the rate-limiting enzyme in heme synthesis. It is inducible by drugs and toxins and is feedback regulated by heme. This unit describes a radiometric assay using [¹4C]succinate as a substrate and a colorimetric assay based on the conversion of ALA to a pyrrole.

Comparison of the beluga whale (Delphinapterus leucas) expressed genes for 5-aminolevulinate synthase with those in other vertebrates.

The cDNA and inferred amino acid sequences were determined for beluga whale (Delphinapterus leucas) erythroid (E) and housekeeping (H) forms of 5-aminolevulinate synthase (ALS), and they were compared with known sequences for five other vertebrates with particular attention to regulatory features. The cDNAs for whale ALS-E and -H encode, respectively, proteins of 582 and 640 amino acids. Sequence alignments suggest that the whale ALS-H, like those for rat and chicken, has an N-terminal mitochondrial targeting sequence of 56 amino acids. There is a high degree of amino acid conservation between the beluga whale proteins and those of other vertebrates, including regulatory elements and functional residues that have been defined in other ALSs. Both whale proteins contain three heme regulatory motifs suggesting that mitochondrial uptake may be regulated by heme. The ALS-E mRNA contains an iron responsive element in its 5'-untranslated region indicating that its expression may be post-transcriptionally regulated by cellular iron. This extensive structural similarity and the presence of the same regulatory elements found in other ALSs indicate that regulation of ALS in beluga whale is similar to that in other vertebrates.

Phylogenetic analysis of the 5-aminolevulinate synthase gene.

The evolution of 5-aminolevulinate synthase (ALS) was studied by acquiring sequence data and generating phylogenetic trees. Gene sequences were already available for a variety of vertebrates (which have both a housekeeping and an erythroid form of the gene), fungi, alpha-proteobacteria, and one protist and one protostome. In order to generate representative trees, ALS sequence data were acquired from various deuterostomes and protostomes. The species and tissues selected for study were beluga whale liver, hagfish blood, sea urchin gonadal tissue, cuttlefish hepatopancreas, horseshoe crab hepatopancreas, and bloodworm blood. The new sequences and those previously published were examined for the presence of heme-regulatory motifs (HRMs) and iron-responsive elements (IREs). The HRMs are present in almost all eukaryotic species, which suggests their fundamental role in the regulation of ALS. The IREs are present in all vertebrate erythroid forms of ALS, which indicates that in those animals, expression of the erythroid form of the enzyme and, hence, hemoglobin production can be influenced by the intracellular content of iron. The new sequences were aligned with previously reported ALS sequences, and phylogenetic analyses were performed. The resulting trees provided evidence regarding the timing of the gene duplication event that led to the two forms of the ALS gene in vertebrates. It appears that the housekeeping and erythroid forms of ALS probably arose before the divergence of hagfish from the deuterostome line leading to the vertebrates. The data also add to the evidence indicating that alpha-proteobacteria are the nearest contemporary relatives of mitochondria.

Purification and properties of periplasmic 3':5'-cyclic nucleotide phosphodiesterase. A novel zinc-containing enzyme from the marine symbiotic bacterium Vibrio fischeri.

The 3':5'-cyclic nucleotide phosphodiesterase (CNP) of Vibrio fischeri, due to its unusual location in the periplasm, allows this symbiotic bacterium to utilize extracellular 3':5'-cyclic nucleotides (e.g. cAMP) as sole sources of carbon and energy, nitrogen, and phosphorus for growth. The enzyme was purified to apparent homogeneity by a four-step procedure: chloroform shock, ammonium sulfate precipitation, and chromotography on DEAE-Sephacel and Cibacron Blue 3GA-agarose. The active enzyme consists of a single polypeptide with a mass of 34 kDa. At 25 degrees C, it has a pH optimum of 8.25, a Km for cAMP of 73 microns, and a Vmax of 3700 mumol of cAMP hydrolyzed/min/mg protein (turnover number of 1.24 x 10(5)/min). The specific activity of the V. fischeri enzyme is approximately 20-fold greater than that of any previously characterized CNP when comparisons of activity are made at the same assay temperature. Activity increases with temperature up to 60 degrees C. The CNP contains 2 atoms of zinc/monomer, and zinc, copper, magnesium, and calcium can restore activity of the apoenzyme to varying degrees. The exceptional specific activity of the enzyme and its unusual location in the periplasm support proposals that the enzyme enables the bacterium to scavenge 3':5'-cyclic nucleotides in seawater and that the enzyme plays a role in cAMP-mediated host-symbiont interactions.

Identification of cytochrome P-450 1A (CYP1A) genes from two teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and phylogenetic analysis of CYP1A genes.

Cytochrome P-450-mediated responses to environmental challenges are well known in diverse animal taxa, but the evolution of the complex gene superfamily coding for these enzymes is poorly understood. Here we report a phylogenetic analysis of the cytochrome P-450 1A (CYP1A) genes including two new sequences determined from teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops). Degenerate PCR primers were used to amplify a 1.2 kbp fragment from liver cDNA. The toadfish PCR product was used as a probe to identify a full-length CYP1A clone from a toadfish liver cDNA library. The entire coding region of the scup CYP1A was obtained by rapid amplification of cDNA ends (RACE) using specific primers based on the sequence of the partial PCR product. The predicted protein sequences for toadfish and scup CYP1A shared 78% and 83% amino acid identity with rainbow trout CYP1A1 respectively. Amino acid identity with mammalian CYP1A proteins ranged from 51 to 60% for 505 aligned positions. Phylogenetic analysis of four teleost fish CYP1A genes (trout, toadfish, scup and plaice) and 12 mammalian CYP1A genes suggests a monophyletic origin of the teleost genes, with the trout gene being most divergent, and indicates three distinct groupings: mammalian 1A1, mammalian 1A2, and fish 1A. This supports the idea that the gene duplication event which gave rise to CYP1A1 and CYP1A2 occurred after the divergence of the lines leading to mammals and fish. These results establish a molecular phylogeny within the CYP1A subfamily, the first such detailed phylogenetic analysis within a cytochrome P-450 family.

Induction of rat hepatic microsomal drug metabolizing enzymes by pyrazole and 4-substituted pyrazoles.

Pyrazole and 4-methylpyrazole are potent inhibitors of liver alcohol dehydrogenase and as such have been proposed as potential antidotes to alcohol poisoning. These drugs are also inducers of hepatic cytochrome P-450. We tested pyrazole and four 4-substituted pyrazoles for their potential as inducers of cytochrome P-450 and drug metabolism in mature male rats. Total cytochrome P-450 was significantly increased (p less than 0.05) 1.3 fold by treatment with 4-methylpyrazole. P-nitrophenol hydroxylase (PNPH) activity (nmol/min/mg protein) was increased 1.9 fold following treatment with pyrazole and with 4-methylpyrazole. Treatment with 4-methylpyrazole also resulted in a 2.9 fold increase in ethoxyresorufin demethylase (EROD) activity. In addition, pyrazole treatment led to a significant decrease in the activity of benzphetamine demethylase. 4-Iodopyrazole increased the turnover (nmol/min/nmol P-450) of EROD and PNPH by 1.5 fold each. 4-Nitropyrazole had no significant effect on any of the activities or turnover rates tested. In contrast to results with cultured chick hepatocytes, where induction was directly related to the hydrophobicity of the 4-substituent, the present data indicate that the process of induction of in vivo is more complex.

Pyrazoles as effectors of ethanol oxidizing enzymes and inducers of cytochrome P450.

The effectiveness of pyrazoles acting as inhibitors of alcohol dehydrogenase in vitro or of ethanol metabolism by intact, isolated hepatocytes is influenced both by the hydrophobicity of the pyrazole and by the electronic properties of the substituents at the 4-position of the pyrazole ring. In contrast, the binding of pyrazoles to cytochrome P450 in vitro and the induction of P450(s) in cultured hepatocytes are dependent only on hydrophobicity. The high correlation between the binding of pyrazoles in vitro and their ability to induce P450(s) in cultured liver cells suggests as a working hypothesis that the pyrazole:P450 complex has a role in the induction process.

Subcellular location of phosphoenolpyruvate carboxykinase in hepatocytes from fed and starved rats.

To evaluate published indications that about 25% of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), is located in mitochondria of adult rat liver, cell fractionations were conducted with hepatocytes isolated from rats that were fed ad libitum or starved for 2 days. Hepatocytes were exposed to digitonin for 10 s, and the released materials were separated from residual cell structures by centrifugation through a layer of brominated hydrocarbon. In addition to PEPCK, activities of 9 other enzymes were measured in the untreated cells and with good recovery in the two fractions obtained with digitonin treatment. By comparison with the release of marker enzymes for the cytosol and mitochondria, the subcellular distribution of PEPCK was determined. With cells from either fed or 2-day-starved rats, this enzyme was released exactly like lactate dehydrogenase and within 2-3% of phosphoglycerate kinase and pyruvate kinase. These results indicate that, even after induction by starvation, at least 97% of PEPCK activity is located in the cytosol of rat liver.

A quantitative structure-activity relationship and molecular graphics analysis of hydrophobic effects in the interactions of inhibitors with alcohol dehydrogenase.

An analysis of the inhibition constants of pyrazoles, phenylacetamides, formylbenzylamines, and acetamides acting on liver alcohol dehydrogenase (ADH) yields quantitative structure-activity relationships (QSAR) having a linear dependency on octanol-water partition coefficients (log P). The average coefficient and standard deviation with the log P term for six different QSAR is 0.96 (+/- 0.14). This suggests complete desolvation of the substituents (directly comparable to partitioning into octanol) on binding to the enzyme. Study of a molecular graphics model of ADH constructed from the X-ray crystallographic coordinates shows that the substituents are engulfed in a long hydrophobic channel which is so narrow that water of solvation must be removed from them in the binding process.

Induction of cytochrome P-450 by alcohols and 4-substituted pyrazoles. Comparison of structure-activity relationships.

A comparison was made between 4-substituted pyrazoles and short-chain alcohols as inducers of cytochrome P-450. A quantitative structure-activity analysis of the data led to the following equations: (I) Pyrazoles: Log 1/C = 0.85 (+/- 0.21) Log P + 1.93 (+/- 0.38), r = 0.970 (II) Alcohols: Log 1/C = 0.78 (+/- 0.14) Log P + 1.46 (+/- 0.13), r = 0.988 where C is the concentration that caused a 50% increase in cytochrome P-450, is the partition coefficient between octanol and water, and r is the correlation coefficient. The results suggest that induction of cytochrome P-450 by these compounds depends on hydrophobicity alone. Electronic and steric factors have insignificant roles.

Metabolite and enzyme contents of freeze-clamped liver of the marine fish Stenotomus chrysops.

Hepatic metabolites and enzymes in the marine fish, scup or porgy (Stenotomus chrysops), were determined in freeze-clamped tissue taken either within a day of removing fish from their natural habitat or after scup were held in captivity for 6-8 months. The same determinations were made for liver from fed or 48 hr-starved rats (Mus norvegicus albinus). Compared with rat liver, both groups of fish had, per gram of liver, higher contents of AMP, inorganic phosphate, glucose, glucose-6-phosphate, malate, glutamate and NH4+. ATP was lower in fish liver, and ADP, lactate and pyruvate contents were similar in rats and fish. Fish held in captivity had significantly lower pyruvate, alpha-ketoglutarate, and cytosolic free NAD+/NADH and higher cytosolic free NADPH/NADP+. These decreases were similar to those seen when starved rats were compared with fed ones. In scup liver, glucose-6-phosphate dehydrogenase was 3-8 times, malic enzyme about 2 times, and alanine aminotransferase 2-4 times higher than those activities in rat liver. Those results and a higher cytosolic free NADPH/NADP+ are consistent with the liver being the major site of lipogenesis in fish.

Compartmentation of enzymes: ATP citrate lyase in hepatocytes from fed or fasted rats.

Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase (EC 4.1.3.8) illustrate the information that uniquely can be obtained. Although the enzyme was previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were starved for 2 days, fed NIH stock diet ab libitum, or starved for 2 days and then refed a fat-free diet for 2 days, the noncytosolic activity was, respectively, 52, 21, or 24% of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 microM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis.

Digitonin perfusion of rat liver. A new approach in the study of intra-acinar and intracellular compartmentation in the liver.

Perfusing a rat liver with digitonin in the concentration range 2-20 mg/ml results in complete decolorization of the organ within 45-250 s. Decolorization progresses with time in the direction of flow, and it is therefore possible, by collecting the eluate, to obtain material from specific intracellular compartments of hepatocytes in different zones in the microcirculatory unit of the liver. The results demonstrate that cytoplasmic marker enzymes from periportal or perivenous hepatocytes can be collected with as little contamination from the other compartment as is obtained in micro-dissection studies. Furthermore, a fraction enriched in mitochondrial marker enzymes can be achieved with only 10-20% contamination by cytoplasmic material.

Selective inhibition of alanine aminotransferase and aspartate aminotransferase in rat hepatocytes.

Experiments were conducted with intact rat hepatocytes to identify inhibitors and incubation conditions that cause selective inhibition of alanine aminotransferase or aspartate aminotransferase. Satisfactory results were obtained by preincubating cells with L-cycloserine or L-2-amino-4-methoxy-trans-but-3-enoic acid in the absence of added substrates. When cells were incubated for 20 min with 50 microM-L-cycloserine, alanine aminotransferase activity was decreased by 90%, whereas aspartate aminotransferase was inhibited by 10% or less. On subsequent incubation, synthesis of glucose and urea from alanine was strongly inhibited, but glucose synthesis from lactate was unaffected. L-2-Amino-4-methoxy-trans-but-3-enoic acid (400 microM) in hepatocyte incubations caused 90-95% inactivation of aspartate aminotransferase, but only 15-30% loss of alanine aminotransferase activity. After preincubation with the inhibitor, glucose synthesis from lactate was almost completely blocked; with alanine as the substrate, gluconeogenesis was unaffected, and urea synthesis was only slightly decreased. By comparison with preincubation with inhibitors, simultaneous addition of substrates (alanine; lactate plus lysine) and inhibitors (cycloserine; aminomethoxybutenoic acid) resulted in smaller decreases in aminotransferase activities and in metabolic rates. Other compounds were less satisfactory as selective inhibitors. Ethylhydrazinoacetate inactivated the two aminotransferases to similar extents. Vinylglycine was almost equally effective in blocking the two enzymes in vitro, but was a very weak inhibitor when used with intact cells. Concentrations of DL-propargylglycine (4 mM) required to cause at least 90% inhibition of alanine aminotransferase in hepatocytes also caused a 16% decrease in aspartate aminotransferase. When tested in vitro, alanine aminotransferase was, as previously reported by others, more sensitive to inhibition by amino-oxyacetate than was aspartate aminotransferase, but in liver cell incubations the latter enzyme was more rapidly inactivated by amino-oxyacetate.

The inhibition of alcohol dehydrogenase in vitro and in isolated hepatocytes by 4-substituted pyrazoles.

As a means of comparing the functional properties of an enzyme in dilute solution in vitro with those for the same enzyme acting in its normal cellular environment, a study was conducted with 4-substituted pyrazoles as inhibitors of rat liver alcohol dehydrogenase in vitro and ethanol oxidation in isolated rat hepatocytes. Inhibitor constants (Ki's) for the same set of pyrazole derivatives were also determined for human liver alcohol dehydrogenase. The best-fitting equations were derived to relate the Ki's to the chemical nature of substituents. These quantitative structure-activity relationships show that pyrazoles with stronger electron-withdrawing substituents are weaker inhibitors both for the enzyme in vitro and, to an equal extent, for ethanol oxidation by intact cells. Inhibitor effectiveness is also dependent on substituent hydrophobicity, but, while increasing hydrophobicity makes stronger inhibitors of the enzyme in vitro, it can diminish the effectiveness in vivo by decreasing permeability through the cell membrane. A structure-activity analysis of published Ki's for pyrazoles acting against human pi-ADH indicates that its active site differs from those in other alcohol dehydrogenases.

Enzymatic measurement of ethanol or NAD in acid extracts of biological samples.

An enzymatic method for the measurement of ethanol has been developed to permit analyses with unneutralized acid extracts of blood, liver, cell suspensions, or other biological materials. Components of the assay mixture include NAD, yeast alcohol dehydrogenase, tris(hydroxymethyl)aminomethane (Tris), and lysine. Tris is a trapping agent for the reaction product, acetaldehyde. Lysine is used to maintain the pH at 9.7 where oxidation of ethanol is quantitative and most rapid, even when as much as 0.2 ml of 0.5 N HClO4 is added. Lysine also causes the reaction to be 2 to 4 times faster than it is when either glycine or 2-amino-2-methyl-1-propanol is used as the buffer. The assay is linear up to an ethanol concentration of 0.125 mM in the reaction mixture and is complete by 4 min. By substituting ethanol for NAD in the reagents, the assay performs equally well in measuring NAD.