Studies on the production and assessment of experimental histidinemia in the rat

Intraperitoneal administration to rats of D- or DL-α-hydrazunoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lysase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize l-[ring-2-¹⁴C] histidine to ¹⁴CO₂. Rats in which hepatic histadine ammonia-lyase activity was either depressed by dl-hydrazunoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of ³H₂O release into plasma water following l-[3-H]histidine administration. Plasma l-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of dl-α-hydrazinoimisazolyl-proprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the dl-α-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in l-histidine degradation in the rat. The potential usefulness of dl-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.     

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.     

Histidine ammonia-lyase from rat liver. Purification, properties, and inhibition by substrate analogues.

Histidine ammonia-lyase (EC 4.3.1.3) from rat liver was purified more than 250-fold to near homogeneity. Electrophoretic determinations indicated a native molecular weight of approximately 200,000. The enzyme has a pH optimum of approximately pH 8.5. The minimum Km for L-histidine was 0.5 mM at pH 9.0. The Michaelis constant in the physiological pH range was, however, more than 2.0 mM. D-alpha-hydrazinoimidazolylpropionic acid was found to be a potent competitive inhibitor of liver histidine ammonia-lyase (Kis=75 muM); the L enantiomer of this compound was less effective in this regard. The enzyme was also inhibited competitively by L-histidine hydroxamate (Kis=0.4 mM), and to a lesser extent by L-histidinol, D-histidine, and glycine. Failure of a wide variety of other histidine analogues to inhibit the enzyme substantially indicates high specificity of the active site for L-histidine. No alternate substrates were identified for the enzyme. DL-alpha-Hydrazinophenylpropionic acid, the alpha-hydrzino analogue of phenylalanine, was similarly shown to be a very potent competitive inhibitor of a mechanistically similar L-phenylalanine ammonia-lyase purified from Rhodotorula glutinis. The properties of histidine ammonia-lyase from rat liver differ significantly from those of the enzyme from Pseudomonas fluorescens which has been studied most extensively to date.     

Effect of treatment with hemin on rat liver catalase.

Rats were injected with a single or repeated doses of hemin intraperitoneally, and the effect on liver catalase [EC 1.11.1.6] was studied. A single administration of hemin caused a reduction in the concentration of liver catalase, both in enzymatic activity and in catalase protein determined immunochemically. The reduction occurred a few hours after the hemin injection, and is probably due to stimulated degradation. Disappearance of radioactivity from liver catalase prelabelled with [14C]leucine was enhanced following the administration of hemin. No evidence for a repression in vivo incorporation of [14C]leucine and [3H]sigma-aminolevulinic acid into liver catalase was obtained with hemin-treated rats. When the hemin was given repeatedly at 12-h intervals, the level of liver catalase decreased considerably. However, the impairment in catalase-synthesizing activity of liver cells of rats thus treated was rather slight, when examined in a cell-free system. Some differences were noted between the results in the present study and those in previous investigations with Sedormid-treated rats.     

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions.

1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.     

Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

Biosynthesis of liver catalase in rats treated with allylisopropylacetylcarbamide. II. Double-labeling of catalase with (14C)leucine and delta-(3H)aminolevulinic acid.

Double-labeling of liver catalase [EC 1.11.1.6] with [14-c]leucine and delta-[3H]aminolevulinic acid was carried out both in vivo and in vitro using rats treated with allylisopropylacetylcarbamide (Sedormid). These radioactive precursors were incorporated into catalase at a lower rate than in normal rats. In particular, the incorporation of 3H was remarkably inhibited. The results suggest that the administration of Sedormid can inhibit synthesis of the protein moiety of catalase, and possibly interfere with the binding of heme to the catalase protein.     

Use of 3H and 14C doubly labeled glucose and amino acids in the study of hormonal regulation of gluconeogenesis in rats.

Double isotope procedures (3H and 14C) were used in vivo to investigate a) slow long-term gluconeogenic actions of adrenal glucocorticoids, and b) rapid stimulation of gluconeogenesis by glucagon. [U-14C,6-3H]Glucose was administered to normal and adrenalectomized rats. No effect was observed on the [6-3H]glucose half-life suggesting the dicarboxylic acid shuttle is unaffected by adrenalectomy; the Cori cycle is also not influenced. Loads of [14C]aspartate, [14C]glutamate, or [14C]alanine were given to normal and adrenalectomized rats. Simultaneously, in vivo transaminase activity was studied by measuring the appearance of 3H2O in body water after administration of [2-3H]aspartate, [2-3H]glutamate, or [2-3H]alanine, Adrenalectomy has no influence on the incorporation of glutamate or aspartate into glucose or on their in vivo transaminases. Diminution of incorporation of [14C]alanine into glucose and alanine transaminase activities occurs only when rats are given unphysiological loads. These studies support the contention that glucocorticoid rate-limiting actions occur in extrahepatic tissues to produce an increased flow of glucose precursors to the liver. [U-14C,3-3H]Glucose was used to investigate the effect of glucagon on the hepatic fructose-6-phosphate (F-6-P) cycle. Glucagon administration resulted in a rapid drop in the 3H/14C ratio of circulating glucose, suggesting an increase in F-6-P recycling caused by activation of FDPase with little or no decrease in phosphofructokinase. Such a change would direct substrate flux toward gluconeogenesis.     

Metabolism of chylomicron arachidonic and linoleic acid in the rat

Chyle and chylomicrons, obtained after feeding thoracic duct cannulated rats [3H]arachidonic (20:4) and [14C]linoleic acid (18:2) in cream, were injected i.v. into recipient animals. 7.5-15 min after injection, the 14C/3H ratio of the triacylglycerols remaining in plasma was about half of that in the injected chylomicrons, indicating that the chylomicron remnants formed retained relatively more [3H]20:4 than [14C]18:2. The 14C/3H ratio of plasma diacylglycerols was about 6-fold lower than that of plasma free fatty acids. The proportion of [3H]20:4 found in plasma cholesteryl esters was several-fold higher than that of [14C]18:2. Inhibition of hepatic lipase by a specific antiserum did not significantly influence the clearance of triacylglycerols, but increased the amount of 3H in plasma diacylglycerols. It also prevented the rapid clearance of phosphatidylethanolamine from plasma. The liver uptake of [3H]20:4 exceeded that of [14C]18:2. Antiserum against hepatic lipase diminished the difference. In contrast, the 14C/3H ratio of adipose tissue was higher than that of the injected chyle lipoproteins.     

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Histidine ammonia-lyase. The use of 4-fluorohistidine in identification of the rate-determining step.

The alpha,beta eliminations of NH3 from L-histidine and 4-fluoro-L-histidine by histidine ammonia-lyase appear to occur by similar mechanisms, although a large difference in Vmax for the two reactions was observed. Both reactions were shown to be reversible with an equilibrium constant of 4 to 5. The presteady state kinetics of the deamination of 4-fluoro-L-histidine indicates that the rate-determining step precedes the dissociation of ammonia from the enzyme. The isotope effect of 1.4 to 2.0 observed with 4-fluoro-DL-[beta-2-H2]histidine or DL-[beta-2-H2]histidine or DL-[beta-2-H2]histidine indicates that the C-H bond breakage is at least partially rate-determining for the deamination of both substrates.     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

Mechanism of the hypertriglyceridemia induced by tumor necrosis factor administration to rats

4 h after intravenous injection of recombinant HuTNF-alpha to fed rats, an increase in heart, diaphragm, and plasma lipoprotein lipase activity was observed. At the same time, a 40-60% decrease in enzymic activity in epididymal fat pad and kidney and 40% decrease in hepatic lipase activity in liver had occurred. Similar results were obtained 20 h after injection of recombinant HuTNF-alpha into fasted rats. Pretreatment with Indomethacin did not affect the changes in tissue lipoprotein lipase activity observed following recombinant HuTNF-alpha administration. Serum triacylglycerol concentration increased by 2- and 6-fold; 4 and 20 h after recombinant HuTNF-alpha administration. Disappearance of 14C-labeled triacylglycerol from the circulation after injection of small chylomicrons, biosynthetically labeled in their triacylglycerol and cholesterol moieties, was lower in TNF-treated than in control rats. However, the clearance rate of triacylglycerol was the same or even higher in recombinant HuTNF-alpha treated rats (assuming that 14C-labeled chylomicron triacylglycerol represents the serum triacylglycerol pool). The livers of recombinant HuTNF-alpha-treated rats and controls contained similar amounts of 14C-labeled lipids, but less [3H]cholesterol, suggesting that in recombinant HuTNF-alpha-treated rats, the liver took up chylomicron remnant particles enriched with triacylglycerol. Separation of the d less than 1.04 g/ml fraction of serum obtained from control and recombinant HuTNF-alpha treated rats by zonal ultracentrifugation revealed that in recombinant HuTNF-alpha-treated rats the lipoprotein particles were less lipolyzed than in controls. The secretion rate of [3H]triacylglycerol into the serum was determined 90 min after injection of [3H]palmitate albumin complex and Triton WR 1339. In recombinant HuTNF-alpha-treated rats, the secretion of [3H]triacylglycerol into plasma was 48% higher than in controls. It is suggested that the increase in lipoprotein lipase activity of heart and diaphragm resulted from an indirect effect of TNF. It is concluded that the increase in serum triacylglycerol in the recombinant HuTNF-alpha-treated rats is due mainly to an increased secretion of triacylglycerol by the liver. Impaired lipolysis, probably due to a fall in hepatic lipase could also contribute to the rise in plasma triacylglycerol.     

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

Impaired plasma membrane glycoprotein assembly in the liver following acute ethanol administration

The in vivo effects of acute ethanol administration on hepatic plasma membrane assembly were studied in the rat. When [14C]fucose and [3H]N-acetylmannosamine, a sialic acid precursor, were injected following an acute dose of ethanol, minimal effects on fucose and a slight reduction of sialic acid incorporation into the total pool of hepatic membrane glycoproteins were observed. However, the assembly of labeled fucoproteins and sialoproteins into the plasma membrane was markedly inhibited in the ethanol-treated animals. These results indicate that ethanol administration impairs the late stages of membrane assembly which include the transport of glycoproteins from the Golgi complex to the plasma membrane and/or the insertion of glycoproteins into the membrane.     

Effect of Dexamethasone on Transport of α-Aminoisobutyric Acid and Sucrose Across the Blood-Brain Barrier

The effect of glucocorticoids on the blood-brain barrier (BBB) was studied in rats following a single injection or 3 days of dexamethasone administration. Tracers with a low permeability across the intact endothelium, [14C]sucrose and alpha-[3H]aminoisobutyric acid ([3H]AIB), were simultaneously injected intravenously in untreated rats or in rats treated with dexamethasone. Unidirectional blood-to-brain transfer constants (Ki) in 14 regions of the rat brain were determined. In regions of control brain, average Ki values for AIB and sucrose were approximately 0.0020 and 0.00060 ml g-1 min-1, respectively. The lowest transfer constants were found in caudate nucleus, hippocampus, white matter, and cerebellum. In dexamethasone-treated animals, Ki values for both sucrose and AIB markedly decreased by 30-50% in almost all brain regions. These results indicate that a single injection or 3 days of treatment with dexamethasone causes an apparent reduction in the normal BBB permeability, and dexamethasone may greatly interfere with drug delivery into brain. These observations may have an importance for the administration of drugs in brain disease in the presence of steroids.     

The control of nucleic acid and nicotinamide nucleotide synthesis in regenerating rat liver

1. The effect of injecting nicotinamide on the incorporation of [(14)C]orotate into the hepatic nucleic acids of rats after partial hepatectomy was investigated. 2. At 3h after partial hepatectomy the rapid incorporation of [(14)C]orotate into RNA, and at 20h after partial hepatectomy the incorporation of [(14)C]orotate into both RNA and DNA, were inhibited in a dose-dependent fashion by the previous injection of nicotinamide. 3. The injection of nicotinamide at various times before the injection of [(14)C]orotate at 20h after partial hepatectomy revealed an inhibition of the incorporation of orotate into RNA and DNA which was non-linear with respect to the duration of nicotinamide pretreatment. 4. The induction of a hepatic ATP depletion by ethionine demonstrated that the synthesis of hepatic NAD and NADP in partially hepatectomized rats was more susceptible to an ATP deficiency than in control rats. 5. The total hepatic activity of ribose phosphate pyrophosphokinase (EC 2.7.6.1) was assayed at various times after partial hepatectomy and found to be only marginally greater than the maximum rate of hepatic NAD synthesis induced in vivo by nicotinamide injection between 12 and 24h after partial hepatectomy. 6. It is suggested that a competition exists between NAD synthesis and purine and pyrimidine nucleotide synthesis for available ATP and particularly 5-phosphoribosyl 1-pyrophosphate. In regenerating liver the competition is normally in favour of the synthesis of nucleic acid precursors, at the expense of NAD synthesis. This situation may be reversed by the injection of nicotinamide with a subsequent inhibition of nucleic acid synthesis.     

Biosynthesis of enzymes of peroxisomal beta-oxidation

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for 2 weeks. The increases in the individual enzymes of the hepatic peroxisomal beta-oxidation system after administration of DEHP were 31- to 33-fold. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rates of synthesis of the enzymes were 16- to 20-fold higher and those of degradation were 1.7- to 1.9-fold lower in the DEHP group. The translation rates of these enzymes in vitro with liver RNA in the reticulocyte-lysate system were 12- to 14-fold higher in the DEHP group. Short-term kinetic labeling experiments on acyl-CoA oxidase consisting of three subunits were conducted in vivo to explore the biogenesis of peroxisomes. The label was found in the biggest subunit of the enzyme in the supernatant fraction shortly after the label injection, but was distributed to the smaller subunits later. The labeling in the smaller subunits in the peroxisomal fraction was greater than that of the supernatant. The distribution of the label among the subunits in these subcellular fractions was the same as that of the protein amounts 1 day after the label injection. This paper reports that the increase in the quantities of peroxisomal enzymes upon administration of DEHP is mainly due to the increase in their synthesis rates caused by the increase in amounts of mRNA coding for these enzymes.     

In vivo regulation of histidine ammonia-lyase activity from Streptomyces griseus.

The enzyme histidine ammonia-lyase (histidase) is required for growth of Streptomyces griseus on L-histidine as the sole source of nitrogen. Histidase was induced by the inclusion of histidine in the medium, regardless of the presence of other carbon and nitrogen sources. Histidase activity was increased by a shift of culture incubation temperature from 30 to 37 degrees C. Conversely, upon induction of sporulation by either phosphate starvation or nutritional downshift, histidase underwent rapid inactivation. Nutrient replenishment fully reversed histidase inactivation while simultaneously permitting reinitiation of vegetative growth. In contrast to histidase inactivation during sporulation, histidase was activated after transition of a vegetatively growing culture to stationary phase. Although neither activation nor inactivation required de novo protein synthesis, inactivation appeared to involve a heat-labile protein. The results indicate that histidase activity is regulated in vivo by a process that responds to changes in the growth phase of the organism.