Deamidase Activity Research Articles

Characterization of the deamidase enzyme responsible for the metabolism of the anti-cancer peptide: H-Arg- d-Trp-N mePhe- d-Trp-Leu-Met-NH 2

H-Arg- d-Trp-N mePhe- d-Trp-Leu-Met-NH 2, a broad spectrum neuropeptide growth factor antagonist (antagonist G), is soon to enter a phase I clinical trial for the treatment of small-cell lung cancer (SCLC). The pre-clinical pharmacology of this peptide has revealed that its metabolism proceeds from the C-terminus via deamidation. In this study the class of enzyme responsible for the degradation of antagonist G has been characterized. Tissue distribution studies on the enzyme have shown it to be very widespread with high specific activity being detected in the spleen, kidney, H69 SCLC xenograft and liver (12.64, 9.58, 8.00 and 6.94 nmols G/mg protein/hr, respectively). HPLC gel filtration indicated that the G-dearnidase enzyme had an apparent molecular mass of 81 kDa. The sub-cellular distribution of the enzyme using differential centrifugation indicates that it is largely soluble with >85% of the activity located in the cytosolic fraction. The distribution of activity towards antagonist G closely resembles that of esterase and acid carboxypeptidase activity, two activities, along with deamidase activity, known to be possessed by serine carboxypeptidases. Studies using a range of protease inhibitors showed clear inhibition of metabolism by phenylmethylsulphonylfluoride and benzyloxycarbonyl-phenylalanine chloromethylketone, indicating that the enzyme is a chymotrypsin-like serine carboxypeptidase. This knowledge of the enzyme will be invaluable in the further development of antagonist G and similar compounds. Moreover, the widespread distribution of this enzyme together with its broad specificity for C-terminal group suggests that it should be given serious consideration when designing C-terminally modified peptide drugs.

Plasma membrane-bound and lysosomal peptidases in human alveolar macrophages.

Alveolar macrophages protect the lungs against noxious agents. Proteases and peptidases are essential for this defense and many metabolic activities. Human alveolar macrophages were evaluated for the presence of six important peptidases. Deamidase, a serine peptidase identical with the lysosomal protective protein and possibly with cathepsin A, had high specific activity in alveolar macrophages and is also present in cultured mouse J774A.1 and human U937 cells, used for the sake of comparison. In fractionated J774A cells, most of the deamidase activity was in the lysosomal fraction and in the final supernatant. Deamidase in human alveolar macrophages, obtained by bronchoalveolar lavage from 23 patients, cleaved dansyl-Phe-Leu-Arg at a rate of 2.26 mumol/h/mg protein and hydrolyzed the chemotactic peptide N-f-Met-Leu-Phe even faster, at a rate of 53.1 mumol/h/mg protein, the highest activity for this enzyme with any of the cells we tested. Rabbit antiserum, elicited with the recombinant partial sequence of the enzyme, immunoprecipitated 77-88% of the macrophage deamidase. In immunocytochemistry, this antiserum localized deamidase within the human macrophages. The enzyme was inhibited by diisopropylfluorophosphate (DFP; 1 mM) and by ebelactone B (10 microM), noncompetitively. The mRNA of deamidase was detected in mouse macrophages by Northern blot; the two protein chains of deamidase were shown in human macrophages by Western blot. In addition, two other serine peptidases were also highly active in macrophages: dipeptidyl peptidase IV (1.38 mumol/h/mg protein) and prolylcarboxypeptidase (0.72 mumol/h/mg protein). The activity of plasma membrane zinc metallopeptidases, neutral endopeptidase 24.11 and carboxypeptidase M, in contrast, was low or absent (angiotensin I converting enzyme; kininase II).(ABSTRACT TRUNCATED AT 250 WORDS)

Dominant chymotrypsin-like esterase activity in human lymphocyte granules is mediated by the serine carboxypeptidase called cathepsin A-like protective protein.

We identified a chymotrypsin-like activity in the granules of IL-2 lymphokine-activated killer (LAK) cells and a NK cell line (YT) that reacted preferentially with the oligopeptide substrate succinyl-Phe-Leu-Phe-thiobenzyl ester (Suc-Phe-Leu-Phe-SBzl). The enzyme was isolated by detergent extraction of sedimented cytotoxic granules and then by a sequence of sieve, hydrophobic, and anion exchange chromatography. On SDS-PAGE, the protein migrated at 42 kDa in nonreduced form and became two bands (31 and 19 kDa, respectively) after reduction. Amino-terminal sequencing of the reduced protein bands revealed 100% homology with cathepsin A-like protective protein (CAPP), a lysosomal enzyme that expresses serine carboxypeptidase and deamidase activities. The carboxypeptidase activity of lymphocyte CAPP was verified by showing that the protease preferred hydrophobic amino acids in the penultimate position of the C terminus (i.e., cleaved arginine from dansyl-Phe-Leu-Arg). The presence of lymphocyte CAPP in secretory lysosomes was demonstrated by showing that Suc-Phe-Leu-Phe-SBzl activity co-migrated with tryptase and Asp-ase activities on Percoll density gradients and that 95% of the Suc-Phe-Leu-Phe-SBzl activity in granule fractions of cavitated YT cells could be immunoprecipitated with an anti-CAPP antiserum. In addition, calcium ionophore-stimulated YT cells were shown to secrete immunoprecipitable CAPP. As proposed for platelets, lymphocyte CAPP may be secreted to function extracellularly by inactivating bioactive peptides.

Transport of thyrotropin releasing hormone in rabbit buccal mucosa in vitro.

The transport of thyrotropin releasing hormone (TRH) in rabbit buccal mucosa in vitro has been investigated with respect to (a) rate and type of metabolism of TRH on mucosal and serosal sides of buccal mucosa, (b) mechanism of TRH transport including charge effect on its permeability, and (c) pathway and rate-limiting regions of TRH movement. In addition, the integrity of excised buccal mucosa has been evaluated for purposes of in vitro solute diffusion experiments using tissue ATP level data, transmission electron microscopy, and TRH transport kinetic data. The results indicate that excised rabbit buccal mucosa can be used for TRH diffusion studies for approximately 6 hr. In addition, TRH apparently traverses buccal mucosa by simple diffusion with a steady-state permeability of about 10(-7) cm/sec, and this permeability is independent of pH. Moreover, the primary pathway appears to be via the intercellular space in the rate-limiting barrier, i.e., the upper 50 microns of the epithelium. Finally, TRH is degraded predominantly by deamidase activity, which is followed by, to a lesser degree, carboxypeptidase metabolism.

Galactosialidosis: Simultaneous deficiency of esterase, carboxy-terminal deamidase and acid carboxypeptidase activities

Esterase and deamidase activities at pH 7.0 and carboxypeptidase activity at pH 5.7 were markedly low or deficient in seven galactosialidosis fibroblast strains with deficient activity of “protective protein” for lysosomal β-galactosidase and neuraminidase. No simultaneous deficiency of these three enzyme activities was observed in other lysosomal disease fibroblasts examined in this study. This result strongly suggests that “protective protein” is identical with a multifunctional protein with esterase/deamidase/carboxypeptidase activities and its mutation in galactosialidosis results in deficiency of these three enzyme activities.

A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein".

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.

Pharmacokinetics of pyrazinamide and its metabolites in healthy subjects.

The plasma and urine pharmacokinetic parameters of pyrazinamide and of its metabolites (pyrazinoic acid, 5-hydroxy-pyrazinamide, 5-hydroxy-pyrazinoic acid and pyrazinuric acid) have been studied after a single oral dose of pyrazinamide 27 mg.kg-1 in 9 healthy subjects. Pyrazinamide was rapidly absorbed (tmax less than or equal to 1 h) and showed a short distribution phase followed by an elimination phase of t1/2 beta = 9.6 h. The close similarity of the apparent elimination rates of the metabolites led to a second trial of a single oral dose of pyrazinoic acid to evaluate the formation and elimination stages. The limiting factor was found to be the activity of a microsomal deamidase (pyrazinoic acid formation from pyrazinamide and 5-hydroxy-pyrazinoic acid formation from 5-hydroxy-pyrazinamide). In contrast, oxidation by xanthine oxidase occurred very rapidly (5-hydroxy-pyrazinamide formation and pyrazinoic acid catabolism to 5-hydroxy-pyrazinoic acid).

Purification and characterization of nicotinamide deamidase from yeast.

Nicotinamide deamidase (YNDase) has been purified from yeast through the use of a six-step procedure that includes molecular-sieve high performance liquid chromatography. The final preparation was homogeneous by the criteria of sodium dodecyl sulfate-gel electrophoresis, and the enzyme specific activity was determined to be 175 mumol of nicotinate formed per min/mg enzyme. Gel electrophoresis and molecular-sieve high performance liquid chromatography were employed also to characterize YNDase as a monomeric protein with a molecular weight of 34,000. A Km value for nicotinamide of 33 microM was determined for the deamidase activity at pH 6, and a pH range for optimal stability of 6-8.5 was established for this enzyme. The YNDase activity was also examined over a pH range at several substrate concentrations and both the log Vmax and log Vmax/Km plots versus pH suggested that a protonated amino acid residue with an apparent pKb value of 7.8 was essential to this activity. During an in vitro assay of the YNDase-catalyzed formation of nicotinate, ammonia was generated and detected chemically. Inhibition of the YNDase activity by nicotinaldehyde suggested the presence of either an essential lysine (Schiff's base formation) or cysteine residue (thiohemiacetal intermediate) at the YNDase active site. The relatively large value of the nicotinaldehyde inhibition constant (Ki = 68 microM), the observation that this analogue is a noncompetitive inhibitor of nicotinate formation, and the fact that this inhibition can be rendered irreversible through incubation with sodium borohydride, indicates that a Schiff's base intermediate is more likely to occur upon incubation of YNDase with nicotinaldehyde. However, YNDase is inactivated completely and irreversibly by N-ethylmaleimide at pH 6, and the enzyme is protected against this modification by either nicotinamide or nicotinate. These results suggest that both nicotinate and nicotinamide bind to YNDase, even though the enzymatic reaction is essentially irreversible, and that a cysteine residue may be present at the YNDase active site.

The pyridine nucleotide cycle of Salmonella typhimurium: genetic characterization of the pncXA operon.

A series of Mud1 and Tn10 insertions were identified in the pncA chromosome region of Salmonella typhimurium which is responsible for the production of nicotinamide deamidase. Both pncA (resulting in no nicotinamide deamidase activity) and pncX (resulting in lowered nicotinamide deamidase activity) insertions were constructed. In addition, mutants which could utilize nicotinamide as a sole source of nitrogen were isolated. These mutants, designated pncH, hyperproduce nicotinamide deamidase. Genetic studies utilizing pncX--lacZ and pncA--lacZ operon fusions indicate that pncX::Tn10 insertions reduce transcription of pncA--lac while pncH mutations increase the expression of both pncA--lacZ and pncX-lacZ. The gene order was determined as purB--pncA--pncX--gdh with transcription of both pncA and pncX occurring in the counterclockwise direction. Merodiploid studies suggest a model whereby pncX and pncA form an operon with the major promoter occurring upstream from pncX. A second, weaker promoter for pncA must be situated between pncX and pncA. The pncH mutations appear to occur in the pncX promoter (pncXp) increasing promoter activity.

Thyrotropin-Releasing Hormone Metabolism and Extraction by the Perfused Guinea Pig Placenta*

This report describes the extraction of synthetic TRH and its metabolic conversion in the perfused guinea pig placenta. These studies were performed to obtain an estimate of fractional fetal TRH losses through the placenta and to determine if some of these losses are due to TRH metabolism. The in situ guinea pig placenta was perfused through an umbilical artery for 90 min, and placental effluent fractions were collected at timed intervals from the umbilical vein. Experiments were performed in which the perfusion buffer contained 0.01, 1, and 10 micrograms/ml or no synthetic TRH. Synthetic TRH was always perfused in the presence of 3H2O. In experiments in which TRH was perfused, the perfusion reservoir contents and placental effluent fractions were counted for 3H, and TRH and deamido-TRH were determined by RIA. Similarly, cyclo(His-Pro) was measured when 10 micrograms/ml TRH were perfused. When no TRH was perfused, the perfusion reservoir and placental effluent contents were processed to determine their content of TRH immunoreactivity. When synthetic TRH was perfused, steady state TRH concentrations were achieved in placental effluent fractions by 20-30 min. The single pass extraction of TRH by the placenta was 11.4 +/- 2.6% (mean +/- SE) compared to 56.9 +/- 7.0% for 3H2O (P less than 0.001). No significant difference was detected regardless of whether 10, 1, or 0.01 micrograms/ml TRH were perfused. A portion of the TRH that perfused the placenta was converted to deamido-TRH at all concentrations of perfused TRH. No conversion of TRH to cyclo(His-Pro) was noted when the highest concentration (10 micrograms/ml) of TRH was perfused. The conversion of TRH to TRH-OH was 4.2 +/- 0.7% in a single pass. When the perfusion buffer was devoid of synthetic TRH, a small but significant increase in the content of TRH immunoreactivity was noted in the placental effluent compared to that in the perfusion reservoir. This was not large enough to affect calculations of the placental extraction of TRH. These studies, in addition to demonstrating that the placenta contains TRH deamidase activity, suggest that losses of fetal TRH through the placenta are not large. They do not support the current impression, based on the fetal TSH response to maternal TSH administration, that the placenta is freely permeable to TRH.

Preliminary evidence for a pyridine nucleotide cycle in Bordetella pertussis.

Preliminary evidence that Bordetella pertussis has a functional pyridine nucleotide cycle was the observation that [14C]-nicotinic acid was rapidly metabolized during its uptake by the bacteria to pyridine nucleotides and nicotinamide. Nicotinamide deamidase activity, necessary for the completion of the cycle by conversion of nicotinamide to nicotinic acid, was found in a soluble extract (20 000 X g supernatant) of B. pertussis cell lysates.

Thyrotropin-releasing hormone metabolism in visceral organ homogenates of the rat.

TRH, which is found in a number of sites outside the central nervous system, is degraded by serum and homogenates of brain, liver, and kidney. The presence of TRH deamidase activity in the brain is well documented. In view of the widespread distribution of TRH, the present study was undertaken to evaluate whether TRH deamidase activity is present in visceral organs and to determine the extent to which TRH degradation, as observed under broken cell conditions, can be accounted for by deamidation. Organs from Sprague-Dawley rats were homogenized in 150 mM sodium maleate, pH 7, and aliquots of the 700 X g supernatant were incubated separately with equimolar amounts of synthetic TRH or the deamidated form of TRH, pGlu-His-Pro. The amount of peptide remaining and the conversion of TRH to deamindated TRH in tubes incubated with TRH were measured by specific RIAs at multiple time points. TRH deamidase activity was found in rat liver, kidney, heart, lung, pancreas, ileum, and skeletal muscle. It was time and temperature dependent and was not observed in boiled tissues. Under relative high substrate conditions, approximately 56%, 50%, and 24% of TRH degradation was via the TRH deamidase pathway in liver, kidney, and lung, respectively. In other tissues, the presence of nondeamidase pathways, in addition to the deamidase pathway, could not be ruled out. TRH deamidation accounted for little or no TRH degradation in serum.

Seasonal variation in the accumulation and degradation of thyrotropin-releasing hormone by frog skin in vitro

The possible seasonal variation of the TRH content in the abdominal skin and blood of frogs ( Rana pipiens) was determined. TRH was quantitated by radioimmunoassay after acidic extraction and column chromatography. It was found that there was considerable variability in the TRH content of the skin and blood among individual frogs and there was no seasonal variation in the TRH content in either tissue. Abdominal skins were capable of TRH accumulation and degradation in vitro. Skins from fall frogs accumulated significantly more exogenously added TRH than skin from frogs collected during the remaining months. The majority of the TRH degrading activity could be accounted for by the activity of TRH deamidase. The deamidated products were released to the incubation medium. The rate of TRH degradation was slightly higher in skins from fall frogs than in those from frogs which had been collected during the other seasons.

Effect of L-tryptophan and L-leucine on biosynthesis of niacin-related compounds in Saccharomyces carlsbergensis.

As a model system for investigating the mechanism of the hepatic NAD-lowering effect of leucine in rats, aerobically grown Saccharomyces carlsbergensis was used in this paper. Tryptophan supplementation of the medium doubled total niacin production by S. carlsbergensis. This elevation in total niacin was mainly due to increases in niacin (14 times) and niacinamide nucleotides (2 times). Among nucleotides, the NAD level doubled whereas NADH, NADP and NADPH levels dropped significantly. Simultaneous supplementation of the medium with leucine suppressed the elevation in total and free niacin levels. In the presence of tryptophan, approximately 50% of the total niacin was secreted in the medium in the form of free niacin, while in the presence of both tryptophan and leucine most of the total niacin remained in the cell. The specific activity of quinolinate phosphoribosyltransferase [EC 2.4.2.19] was not affected by supplementation of the medium with tryptophan and/or leucine. In contrast, the specific activity of nicotinamide deamidase [EC 3.5.1.19] increased fivefold in the presence of tryptophan. Simultaneous supplementation of the medium with leucine tryptophan. Simultaneous supplementation of the medium with leucine suppressed the increase in nicotinamide deamidase. Cellular incorporation of tryptophan was not affected by leucine simultaneously added as a supplement to the medium. Leucine did not have any inhibitory effect on total niacin synthesis from 3-hydroxyanthranilate. From the results, a possible mechanism for the inhibitory effect of leucine on the tryptophan-NAD pathway was discussed.

Effects of benserazide, carbidopa and isoniazid administration on tryptophan-nicotinamide nucleotide metabolism in the rat

Weanling rats were maintained on a diet providing marginally adequate amounts of nicotinamide and vitamin B 6, with a considerable excess of tryptophan to allow endogenous synthesis of nicotinamide nucleotides. Groups of animals received Benserazide or Carbidopa at 0.9 mg/kg body wt/day or isoniazid at 1.3 mg/kg body wt/day. All three drugs led to a reduction in the excretion of N 1-methyl nicotinamide (the principal metabolite of nicotinamide nucleotides in the rat) after 2 and 3 days, compared with control animals receiving the same diet. After 7 days, excretion of this metabolite had returned to the control level. All three drugs are potent inhibitors of tryptophan oxygenase, kynureninase and 3-hydroxyanthranilate oxidase, but the effects of administration in vivo were not related to the apparent K i, values determined in vitro. All three drugs also caused an increase in the activity of nicotinamide deamidase, which may lead to increased efficiency of utilization of dietary nicotinamide in response to deficiency, and hence explain the recovery in N 1-methyl nicotinamide excretion towards the end of the experiment. Urinary excretion of tryptophan metabolites (xanthurenic and kynurenic acids and kynurenine) was not as expected. Benserazide and Carbidopa had no significant effect, while isoniazid caused an increase in kynurenine excretion and a decrease in xanthurenic acid excretion. It is suggested that this may be due to a reduced activity of kynurenine hydroxylase, although isoniazid had no effect on the activity of this enzyme after administration for 11 days, or when added in vitro. The results are discussed in relation to the mechanisms of isoniazid-induced pellagra and Benserazide and Carbidopa-induced niacin depletion in man.

Pyridine nucleotide cycle of Salmonella typhimurium: regulation of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase

Nicotinic acid phosphoribosyl transferase (NAPRTase) and nicotinamide deamidase activities from Salmonella typhimurium were examined regarding their regulation by either feedback inhibition or repression mechanisms. The results indicate that neither enzyme is subject to feedback inhbition. Nicotinamide deamidase does not appear to be under repression control. NAPRTase, however, is repressed when cells are grown in minimal medium supplemented with various intermediates of the pyridine nucleotide cycle. The concentration of exogenously supplied pyridine nucleotide necessary to effect repression of NAPRTas was found to be that concentration which will result in a nadA mutant generation time of less than 60 min. Furthermore, the results presented indicate that nicotinamide adenine dinucleotide is the actual corepressor molecule. The analogs 6-aminonicotinic acid and 6-aminonicotinamide were also capable of repressing NAPRTase, but only when an intact pyridine nucleotide cycl permitted conversion to 6-aminonicotinamide adenine dinucleotide. The role of a repressible NAPRTase is discussed in relation to the overall functioning of the pyridine nucleotide cycle.

Physiological and biochemical studies on senescing tap root nodules of soybeans.

Senescence of soybean (Glycine max L. Merr.) tap root nodules was investigated by comparing changes in various physiological and biochemical activities with changes in capacity to fix nitrogen. Field-grown Beeson and Calland varieties of soybeans of various ages were sources of tap root nodules. With both varieties, the number of tap root nodules per plant remained constant between 56 and 86 days after planting but fresh weight, dry weight, and mass of tap root nodules increased duing this period. Nitrogen (C2H2)fixation by attached tap root nodules was maximum on a fresh weight, dry weight, or nitrogen basis about 56 days after planting for either variety. Metabolic activities of bacteroids as measured by carbon dioxide evolution from glucose and succinate did not appear to vary among nodules of different ages. There was also no indication of mobilization or deposition or deposition of iron, molybdenum, calcium, zinc, and nitrate in aging tap root nodules. Nitrate levels in the aerial portion of the plants decreased significantly after the initial decline in acetylene reduction. Nicotinamide deamidase activity in the cytosol and in extracts of bacteroids did not change significantly as tap root nodules aged. However, significant and consistent changes were observed in initial pH values of nodule breis and the initial decline occurred before (Calland) or concurrently (Beeson) with the initial decline of nitrogen fixation.

Effects of cord factor on microsomal enzymes.

Injection of cord factor into mice induced marked suppression of pyrazinamide deamidase and aminopyrine demethylase activities and slight decreases of the contents of hemoproteins of liver microsomes. Pyrazinamide deamidase activity was most significantly and consistently affected, and might be considered a marker of microsomal damage induced by cord factor. Enzymologic properties of pyrazinamide deamidase of cord factor microsomes and control microsomes did not differ qualitatively. They were both similarly enhanced by bovine serum protein Fraction V, and had similar Km (substrate concentration at which the velocity of the reaction is half-maximal) values. In vitro treatment of microsomes with cord factor did not affect microsomal enzymes.

Activation of the de Novo Pathway for Pyridine Nucleotide Biosynthesis Prior to Ricinine Biosynthesis in Castor Beans

The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de novo pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively.

Nicotinamide deamidase from rabbit liver. III. Inhibition and sedimentation studies

Treatment of rabbit liver nicotinamide deamidase with diisopropyl fluorophosphate or carbobenzoxyamide-2-phenylethyl chloromethyl ketone led to the loss of deamidase activity. In the process, two moles of reagent were covalently bound to the enzyme. Photo-oxidation of two histidine residues also led to the loss of deamidase activity. Acylated enzyme has been implicated as an intermediate in the hydrolysis of nicotinamide by the finding that nicotinamide hydroxyamate was formed when the reaction was carried out in the presence of hydroxylamine. These data are compatible with the characterization of the deamidase as a serine esterase. Guanidinium hydrochloride and sodium dodecyl sulfate, but not urea, caused the enzyme to dissociate. The molecular weight of the subunits obtained on dissociation with sodium dodecyl sulfate was shown to be 60,000 by electrophoresis in polyacrylamide gels. Nicotinamide deamidase was 70% inhibited by l-thyroxine at a molar ratio of thyroxine to enzyme of 2:1. Thyroxine caused the enzymes to enter into a rapid equilibrium involving dissociation into two subunits. The endogenous inhibitor of rabbit liver nicotinamide deamidase was found to be associated with the fatty-acid fraction and to have comparable effectiveness to equivalent weights of purified fatty acids.