Author index for volume 102

A rapid ion exchange chromatographic method was developed for the separation of tissue ornithine from other amino acids, except lysine, interfering with Chinard's ninhydrin method. The reaction was modified to estimate ornithine in the presence of lysine by reducing the color development time to 20 min and by benzene extraction of the chromogen formed. Chromogens of ornithine and lysine have $\text{520}\text{430}\text{nm}$ absorbance ratios of 4 and 1,3, respectively, and this was exploited to correct for lysine interference.     

Developmental and other characteristics of lysine uptake by rat brain synaptosomes

Synaptosomes isolated from adult or newborn rat cerebrum take up l-lysine by two saturable systems, one with a high affinity low capacity and the other with a low affinity high capacity. Initial rate of uptake for low lysine concentrations is more rapid in newborn, but for high concentrations the rate is greater in adult tissue. Analysis of kinetic data indicates that synaptosomes of the newborn have a higher $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ than those of the adult for high affinity system but adult synaptosomes have a higher $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ than newborn for low affinity system. At a physiological lysine concentration of 0.5 mM, the calculated contributions of two systems indicate that the adult uptake occurs for about 71% by low affinity system but the newborn utilizes both systems to the same extent. The uptake is sodium independent but pH dependent. Lysine uptake is inhibited by other dibasic amino acids, arginine and ornithine but not cystine. Kinetic analysis indicates that arginine specifically inhibits the high affinity, low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ system for lysine uptake.     

Synthesis and accumulation of polyamines in rat liver regenerating after treatment with carbon tetrachloride

A single intraperitoneal injection of carbon tetrachloride into rats resulted within 12 hours in a marked accumulation of putrescine in liver with a concomitant decrease in the concentration of spermidine. The accumulation of putrescine apparently was partly due to an immense stimulation of ornithine decarboxylase activity occurring at the same time. However, in addition it was found that during the maximal accumulation of putrescine there was a marked incorporation of radioactivity from labelled spermidine to liver putrescine $\text{in vivo}$. The conversion of spermidine to liver putrescine was hardly detectable in control animals. Besides the treatment with carbon tetrachloride, increased conversion of radioactive spermidine to liver putrescine $\text{in vivo}$ also occurred after treatment with growth hormone, after partial hepatectomy and after treatment with thioacetamide, $\text{i. e.}$ under circumstances characterized by a stimulation of ornithine decarboxylase activity and an increased accumulation of putrescine.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activtivity in rat kidney.Adminsitration of diaminopropane 60 min before partial hepatectomy only marginally inhibited orthine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant.An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy.Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals.Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life.Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [¹⁴C] methionine 4 h after hepatectomy or after administration of porcine growth hormone.Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornitine decarboxylase activity and spemedicine synthesis.     

Plasma arginine and ornithine levels and selected enzyme activities in uremic rats fed various amounts of arginine

Rats weighing 100 g were made chronically uremic by partial left renal artery ligation and contralateral nephrectomy. Rats with urea clearances below 0.30 ml/min and sham-operated controls were pair-fed arginine-free diets, diets containing normal amounts of arginine or diets with high levels of arginine. After 4 to 8 weeks, rats were killed and plasma levels of arginine, ornithine and lysine were measured. In addition, activities of various urea cycle enzymes in liver and kidney and renal transamidinase were determined. Plasma amino acid levels and enzyme activities of the urea cycle remained constant in control rats fed diets differing in arginine content. However, renal transamidinase activity was elevated in control rats fed arginine-free diets. In plasma of uremic as compared with control rats, arginine levels varied with the arginine intake, and lysine levels were elevated when arginine supplements were fed. With all diets, plasma ornithine remained constant in uremic rats at slightly but not significantly increased levels. Hepatic carbamoyl phosphate synthetase activity and renal arginine synthetase activity were reduced in uremic as compared to control rats. Renal transamidinase activity, expressed per g of kidney, was elevated in uremic rats with all diets except arginine-free. When amino acid diets were fed, hepatic arginase activity was higher in uremic rats and this increase was enhanced by arginine-free diets. Other enzyme activities in uremic rats were not affected by the amount of arginine in the diet.     

Relationship between polyamine contents and protease activity in the rat submaxillary gland

The effect of polyamines on the protease activity in the submaxillary gland of castrated rats has been investigated in vivo. The protease activity, which is increased by testosterone, is also increased to a lesser degree by the subcutaneous administration of spermidine. The administration of putrescine was less effective than that of spermidine. The increase of polyamine contents in the submaxillary gland of the castrated rats administered either testosterone or spermidine was nearly parallel to the increase of the enzymatic activity. The administration of methylglyoxal bis(guanylhydrazone), a potent inhibitor of spermidine synthesis, with testosterone inhibited slightly the increase of the protease activity by testosterone, while the administration of the inhibitor with spermidine had essentially no effect on the increase of the enzymatic activity by spermidine. The administration of testosterone also caused a slight increase of $\text{S-}\text{adenosyl-}\text{L}\text{-menthionine}$ decarboxylase activity. These results suggest that spermidine synthesis may be necessary for the stimulation by testosterone of protease synthesis in the rat submaxillary gland.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.     

Inhibition of phorbol ester-induced polyamine accumulation in mouse epidermis by anti-inflammatory steroid

Application of the tumor-promoting phorbol diester 12-$\text{O}$-tetradecanoylphorbol-13-acetate to mouse epidermis causes a large increase in the activity of ornithine decarboxylase and in polyamine accumulation. Concurrent application of fluocinolone acetonide, an anti-inflammatory steroid that dramatically inhibits tumor promotion, resulted in a dose-dependent decrease in the 12-$\text{O}$-tetradecanoylphorbol-13-acetate-stimulated ornithine decarboxylase activity and the subsequent rise in spermidine levels. Spermine and putrescine levels were not greatly affected by fluocinoline acetonide treatment except that maximal putrescine values occurred later in time. Doses of the glucocorticoid as low as 0.1 μg inhibited the 12-$\text{O}$-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity by as much as 50% and the rise in spermidine accumulation by 30% after coincident treatment of female Sencar mice.     

Release of the chloride-dependent arginine aminopeptidase from PMN leukocytes and macrophaces during phagocytosis

The zymosan particles induced a time-dependent release of the chloride-dependent arginine aminopeptidase from rat peritoneal macrophages during $\text{in}$$\text{vitro}$ incubations. Intraperitoneal injections of zymosan, a streptococcal cell preparation and a $\text{Micrococcu}$-suspension caused the release of the chloride-activated arginine aminopeptidase into the peritoneal fluid. The arginine aminopeptidases obtained both from the cell cultivation media and the peritoneal washes were partly purified. The enzymes were similar with regard to the following properties: chloride activation with an optimum at physiological concentrations; strong inhibition by 10^?6M $\text{p}$-chloromercuribenzoate; similar elution properties and preferential hydrolysis of mainly the $\text{N}$-L-aminoacyl-2-naphthylamines of arginine and lysine. The chloride-activated arginine aminopeptidase released into the media in $\text{in}$$\text{vitro}$ conditions was inactivated in contrast to the enzyme released into the peritoneal fluid as a result of the intraperitoneal injections. The timing of the release of the chloride-activated arginine aminopeptidase both $\text{in}$ and $\text{in}$$\text{vitro}$ suggests that the enzyme plays a role in the initial phases of inflammation.     

Control of catabolism of mitochondria in oncogenesis. I. Increase of hepatic mitochondrial population during feeding inactive or marginally active amino azo dyes: absence of de novo biosynthesis

The mechanism of the well established phenomenon that the number of liver mitochondria increases during administration of 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) has been investigated. Fed to rats, both 2-Me-DAB (0.06%) and 4-diethylaminoazobenzene (4-DEAB) (0.0635 %) increase the amount of liver mitochondria by 47% and 31%, respectively. It was established that this is not due to de novo mitochondriogenesis. The increase in the amount of mitochondria correlates with an ~ 10% decrease of total liver protein per g of tissue. Mitochondrial ATP synthesis, which is a prerequisite of any anabolic situation, is drastically impaired following feeding of 2-Me-DAB beyond 1 week as indicated by a very substantial decrease of State 3 respiration, the respiratory control index, and the $\text{ADP}\text{O}$ ratio. Determination of the polysome profile and polysome/monosome ratio at intervals during 2-Me-DAB administration showed no change, despite the fact that mitochondrial components are coded for in both nuclear and mitochondrial DNA. During 4-DEAB administration there was, however, a small but definite rise of the polysome/monosome ratio. Administration of 2-Me-DAB up to 42 days brought about drastic inhibition of the incorporation of [³H]thymidine into both DNA's (approx. 59% with mitochondrial DNA and approx. 77% with nuclear DNA), indicating that these templates could not possibly be involved in the substantial increase of the mitochondrial population. The data suggest that the increase results from a steady accumulation due to increase of the half-life of mitochondria, owing possibly to an inhibition of lysosomal catabolic enzymes.     

Serum glutathione S-transferases: Perinatal development,sex difference,and effect of carbon tetrachloride administration on enzyme activity in the rat

Glutathione $\text{S}$-transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione $\text{S}$-transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione $\text{S}$-transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione $\text{S}$-transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl₄) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.     

Enhancement of hexobarbital-induced sleep by lysine and its metabolites

Lysine has been shown to be metabolized in the rat brain to pipecolic acid which is a precursor of piperidine. Lysine and its proposed metabolites in this pathway were studied for the first time for their effect on the sleeping time induced by hexobarbital in the rat. Only $\text{L}$-lysine and $\text{D}$-lysine were found to prolong sleeping time significantly without toxic effect. A 3-day pretreatment with $\text{L}$-lysine produced an even more profound sleep prolongation. In most cases sleep enhancement was accompanied by a significant shortening of the time of sleep onset. Quantification of brain hexobarbital levels in the control and treated rats indicates that prolongation of sleeping time was not produced by inhibition of hexobarbital metabolism. The sleep prolonging effect of lysine, therefore, may be a direct action of lysine, or the metabolite(s) derived $\text{in}$$\text{vivo}$ from lysine, on the central nervous system.     

Decarboxylation of ornithine and lysine in rat tissues.

The possibility that arginine and lysine might be decarboxylated by rat tissues was investigated. No evidence for decarboxylation of arginine could be found. Lysine decarbosylase (L-lysine carboxy-lyase, EC 4.1.1.18) activity producing CO2 and cadaverine was detected in extracts from rat ventral prostate, androgen-stimulated mouse kidney, regenerating rat liver and livers from rats pretreated with thioacetamide. These tissues all have high ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities. Lysine and ornithine decarboxylase activities were lost to similar extents on inhibition of protein synthesis by cycloheximide and on exposure to alpha-difluoromethylornithine. A highly purified ornithine decarboxylase preparation was able to decarboxylate lysine and the ratio of ornithine to lysine decarboxylase activities was constant throughout purification. Kinetic studies of the purified preparation showed that the V for ornithine was about 4-fold greater than for lysine, but the Km for lysine (9 mM) was 100-times greater than that for ornithine (0.09 mM). These experiments indicate that all of the detectable lysine decarboxylase activity in rat and mouse tissues was due to the action of ornithine decarboxylase and that significant cadaverine production in vivo would occur only when ornithine decarboxylase activity is high and lysine concentrations substantially exceed those of ornithine.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

Rate of synthesis and degradation of rat liver phosphoenolpyruvate carboxykinase in the different thyroid states

The effect of hypo-, eu-, and hyperthyroidism on the relative rate of rat liver phosphoenolpyruvate carboxykinase (PEP-ck) synthesis and degradation was studied in vivo by a radioimmunological technique. In hypo- and euthyroid rats, starvation induced an identical increase in PEP-ck synthesis and activity. Hyperthyroidism led to a significant acceleration in enzyme synthesis and correspondingly enhanced enzyme levels. PEP-ck degradation rate, as measured by the double pulse-labeling technique, was not affected by different thyroid states in starved rats ($\text{t}\text{1}\text{2}\text{~ 8}\text{h}$). Hypo-, eu-, and hyperthyroid starved rats responded to glucose refeeding with a rapid and similar PEP-ck deinduction, followed by a concomitant decrease in enzyme activity.     

L-histidine induced changes in adenosine 3':5'-monophosphate levels in rat brain and amounts of apo-, holo-a and holo-b fatty acid synthetases in rat liver.

When fasted rats were fed a chow or fat-free diet supplemented 5% with L-histidine for three days, the brain adenosine 3′:5′-monophosphate (cAMP) level increased. A 50% increase occurred in rats fed a chow diet and 20% increase in rats fed a fat-free diet. Purification of liver fatty acid synthetase and the isolation of liver apo-, holo-$\text{a}$ and holo-$\text{b}$ fatty acid synthetases demonstrated that L-histidine feeding caused changes in the relative amounts of these enzymes. Apo- and holo-$\text{b}$ fatty acid synthetases increased while the holo-$\text{a}$ form simultaneously decreased. This effect was observed in rats fed either chow or fat-free diets supplemented with L-histidine.     

Increased acridine orange uptake and lysosomal enzyme levels in rat liver after steroid administration

A steroid which stabilizes lysosomes $\text{in vitro}$ and a pyrogenic steroid which labilizes lysosomes $\text{in vitro}$ were compared with respect to their ability to modify lysosomal uptake and lysosomal enzyme levels $\text{in vivo}$. Cortisone acetate increased the uptake of acridine orange by rat liver lysosomes when the dye was administered by intrathoracic injection. The steroid increased and accelerated the uptake of acridine orange so that, in liver lysosomes from treated rats, the maximum uptake was double that of controls and was reached at 2h, whereas in controls the maximum uptake was at 4h after the injection of the dye. This large elevation of uptake is specific to the lysosomal fraction and is not seen in other subcellular fractions of rat liver. The specific activities of a lysosomal enzyme β-N-acetylglucosaminidase were increased in lysosomal fractions from cortisone acetate-treated rats. Etiocholanolone, a steroid which labilizes lysosome $\text{in vitro}$, similarly accelerated and increased acridine orange uptake by lysosomes but had little effect on lysosomal β-N-acetylglucosaminidase levels. Thus the ability of steroids to stabilize or labilize lysosomes $\text{in vitro}$ does not correlate with their effect on lysosomal uptake of injected substances $\text{in vivo}$, or with their ability to induce increased specific activities of lysosomal enzymes.     

Nδ-(phosphonacetyl)-L-ornithine,a potent transition state analogue inhibitor of ornithine carbamoyltransferase

$\text{N}$^δ-(Phosphonacetyl)-L-ornithine, a transition state analogue for the reaction catalyzed by ornithine carbamoyltransferase (EC 2.1.3.3), was synthesized. It strongly inhibited bovine liver ornithine carbamoyltransferase. The inhibition was competitive with respect to carbamoyl-phosphate; the apparent $\text{K}$_m values for carbamoyl-phosphate were 15 μM in 0.05 M $\text{N}$-2-hydroxyethylpiperazine-$\text{N′}$-2-ethanesulfonate (pH 7.2) and 33 μM in 0.1 M Tris-HCl (pH 8.5), and the inhibition constants at pH 7.2 and 8.5 were 7.1 and 4.7 nM, respectively. The inhibition was non-competitive with L-ornithine, the other substrate of the enzyme. This analogue may provide an effective reagent for the elucidation of carbamoyl-phosphate metabolism and its regulation in the liver of ureotelic animals.