Evidence for phospholipid in plasma membrane penicillinase of Bacilluslicheniformis749C

The plasma membrane-bound penicillinase of $\text{Bacillus}$$\text{licheniformis}$$\text{749}\text{C}$ has been purified. Amino acid analysis showed no significant differences in composition between the enzyme and exopenicillinase. Enzyme purified from cultures containing H₃³³PO₄ or [³H]-glycerol contained ³³P or [³H]-glycerol activity and treatment with 8 M urea, 0.2% sodium dodecyl sulfate at 80° C did not remove the ³H-activity from the enzyme protein. Trypsin readily cleaved the glycerol-containing moiety from the enzyme protein, forming enzyme with molecular weight and heat stability like that of the exoenzyme. Phospholipase D and C also produced enzyme resembling the exo-form.     

The effect of protein deficiency on the in vivo binding of aflatoxin B1 to rat liver macromolecules

Male, weanling rats divided into three groups were maintained for 15 days on a semipurified diet containing either 5% casein fed $\text{ad libitum}$ (group 1), 20% casein pair-fed to group 1 (group 2), or 20% casein fed $\text{ad libitum}$ (group 3). Animals on day 16 were injected i.p. with ³H-AFB₁ (1.90 mg/kg) and were sacrificed six hours later. In both the control and protein deficient animals, binding of AFB₁ to DNA was greater than that for chromatin protein. In the protein deficient animals, there was a consistent decrease (70%) in binding to chromatin, DNA and chromatin protein. The decrease in binding to nuclear macromolecules in protein deficient animals is correlated with carcinogenicity and mixed function oxidase (MFO) enzyme activity, and the relationships between carcinogenicity, MFO activity, and binding are discussed.     

Activities of enzymes that metabolize platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in neutrophils and eosinophils from humans and the effect of a calcium ionophore

Enzymatic systems in human blood cells are described for the activation and inactivation of a biologically active phospholipid (1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine) with hypotensive, platelet-aggregating, and inflammatory properties. The results document the presence of alkyldihydroxyacetone-phosphate synthase (forms the $\text{O}$-alkyl linkage in lipids), 1-alkyl-2-lyso-$\text{sn}$-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (produces the biologically active molecule), and 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine: acetylhydrolase (destroys the biological activity) in human neutrophils and eosinophils. Both the acetyltransferase and acetylhydrolase activities are increased severalfold after treatment of normal neutrophils with ionophore A23187; however, alkyldihydroxyacetone-phosphate synthase activity is not influenced by the ionophore. Eosinophils isolated from patients with eosinophilia have significantly greater activities of all the enzymes studied than the eosinophils isolated from normal individuals. Our results indicate the acetyltransferase responsible for 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine synthesis may serve an important role in human blood cells that release this biologically active phospholipid. Moreover, the acetyltransferase activity was found to be dramatically influenced by calcium flux.     

Evidence for ceruloplasmin as a copper transport protein.

Rats fed a copper-deficient diet for eight weeks showed a large decrease in cytochrome $\text{c}$ oxidase in heart, spleen, liver, lung, and pancreas but no significant change in kidney and brain. Three injections of human or rat ceruloplasmin over a five day period greatly increased cytochrome $\text{c}$ oxidase activity in spleen, liver, heart and lung. Rats receiving CuCl₂, Cu-histidine, and Cu-albumin produced a smaller and slower increase in cytochrome $\text{c}$ oxidase compared to ceruloplasmin treated animals. In Cu-histidine treated rats, the increase in enzyme activity did not occur until after the plasma ceruloplasmin level reached a maximal value. It is concluded that ceruloplasmin functions as a primary copper transport protein from which copper atoms are transferred to cytochrome $\text{c}$ oxidase and probably other copper containing proteins.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at $\text{3000 × g}$ followed by sedimentation of a microsomal fraction at $\text{200 000 × g}$. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591–1.1083 were characterized by ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity and [³H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 μm. These fractions contained $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ which appeared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca²⁺ concentration for enzyme activity was 5–10 μM. Both Na⁺ and K⁺ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca²⁺ from the cardiac cell.     

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.     

Crude oil effects on microsomal mixed-function oxidase system components in the striped mullet (Mugil cephalus)

The effects of exposure to two types of crude oil on microsomal mixed-function oxidase system components in livers of juvenile striped mullet ($\text{Mugil cephalus}$) were investigated. Mullet were exposed for 4 days to emulsified Empire Mix or Saudi Arabian crude oils at an initial concentration of 75 ppm and an average of 1 ppm in the water column. Liver size was increased by about 50% following exposure to both oils. Since neither total hepatic protein nor microsomal protein increased as rapidly as did liver size, the concentrations of both were reduced following oil exposures. The proportion of microsomal protein to total hepatic protein or wet weight was not altered following crude oil exposure. Both cytochromes P-450 and $\text{b}$₅ were induced following oil treatment. NADPH-dependent enzymes assayed with cytochrome $\text{c}$ and dichlorophenolindophenol as substrates showed increases in activity after exposure to Empire Mix crude oil but only the latter enzyme activity was increased on a microsomal protein basis following Saudi Arabian crude oil treatment. Activities of NADH cytochrome $\text{c}$ and NADH cytochrome $\text{b}$₅ reductases appeared to vary with the protein level. However, since liver size was increased, oil-treated mullet had more of all parameters measured than did control mullet. Although the acute toxicity of Saudi Arabian crude oil to mullet is greater than that of Empire Mix crude oil, Empire Mix crude oil had greater inductive effects on microsomal oxidase components.     

A high-affinity (Ca2+ + Mg2+)-ATPase in plasma membranes of rat ascites hepatoma AH109A cells

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ had an apparent half saturation constant of $\text{77 ± 31}\text{nM}$ for free calcium, a maximum reaction velocity of $\text{9.9 ± 3.5}\text{nmol}$ ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K⁺, Na⁺, ouabain, KCN, dicyclohexylcarbodiimide and NaN₃, had no significant effect on the activity of high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$. Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol $\text{bis}\text{(β-}\text{aminoethyl ether}\text{)-N,N,N′,N′-}\text{tetraacetic acid}$. Since the kinetic properties of the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ showed a close resemblance to those of erythrocyte plasma membrane $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$, the high-affinity $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.     

Cysteine sulfinic acid decarboxylase in rat brain: Effect of vitamin B6 deficiency on soluble and particulate components

Cysteine sulfinic acid decarboxylase activity is not affected when measured $\text{in vitro}$ in the presence of pyridoxal phosphate in the brains of vitamin B₆-deficient rats. Activity of this enzyme was not detectable in the brains of vitamin B₆-deficient animals when assayed in the absence of pyridoxal phosphate. The activity of cysteine sulfinic acid decarboxylase in crude particulate and soluble fractions of rat brain reflects the distribution of the enzyme $\text{in vivo}$ and the distribution of endogenous B₆ vitamers when assayed in the presence and absence of vitamin B₆. This study indicates that virtually no taurine is synthesized by any component of brain when the animal is subjected to a deficiency of vitamin B₆.     

A Cl−/HCO3−-ATPase in the gils of Carassius Auratus. Its inhibition by thiocyanate

An ATPase is demonstrated in plasma membrane fractions of goldfish gills. This enzyme is stimulated by Cl^? and HCO₃^?, inhibited by SCN^?.Biochemical characterization shows that HCO₃^? stimulation ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.5}\text{mequiv./l}$) is specifically inhibited in a competitive fashion by SCN^? ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.25}\text{mequiv./l}$). The residual Mg²⁺-dependent activity is weakly is weakly affected by SCN^?.In the microsomal fraction chloride stimulation of the enzyme occurs in the presence of HCO₃^? ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{for chloride = 1 mequiv./l}$); no stimulation is observed in the absence of HCO₃^?. Thiocyanate exhibits a mixed type of inhibition ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.06}\text{mequiv./l}$) towards the Cl^? stimulation of the enzyme.Bicarbonate-dependent ATPase from the mitochondrial fraction is stimulated by Cl^?, but this enzyme has a relatively weak affinity for this substrate ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 14}\text{mequiv./l}$).     

Evidence for the involvement of transglutaminase in the uptake of vitellogenin by Xenopuslaevis oöcytes

The yolk protein, vitellogenin, is sequestered by the developing oöcyte by receptor-mediated endocytosis, the process by which cells bind and internalize extracellular proteins. Endocytosis of a variety of proteins follows a similar pathway, whereby internalization of receptor-bound ligand takes place over clathrin-coated regions of the cell membrane. The protein crosslinking enzyme, transglutaminase, has been reported to be essential for the receptor-mediated endocytosis of insulin and α₂-macroglobulin. In this study, the presence of transglutaminase activity was demonstrated in the $\text{Xenopus}\text{laevis}$ ovary and was effectively inhibited by poly L-lysine, an inhibitor of vitellogenin uptake, and dansylcadaverine, a known inhibitor of transglutaminase activity. Two other less poteint inhibitors of transglutaminase, methylamine and bacitracin produced partial inhibition of the ovarian enzyme. Furthermore, dansylcadaverine and methylamine were found to inhibit the appearance of vitellogenin in the yolk platelets of the oöcyte.     

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose

Phospholipase A₂ activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from $\text{E.}$$\text{coli}$ membranes. Phospholipase A₂ activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A₂ activity which was assayable in islet homogenates. Glucose stimulated phospholipase A₂ in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A₂ activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A₂ activity measured in the presence of high calcium concentrations. Phospholipase A₂ activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A₂ activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.     

m-Octopamine as a substrate for monoamine oxidase.

$\text{m}$-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria. The $\text{K}$m and $\text{V}$max values of the brain enzyme were 735 μM and 32.5 nmoles/mg protein/30 min, and those of the liver enzyme 351 μM and 125 nmoles/mg protein/30 min, respectively. The inhibition experiments with clorgyline and deprenyl showed that $\text{m}$-octopamine was a common substrate for type A and type B MAO, though a major part of the activity was due to type A enzyme.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

Limulus anti-LPS factor: An anticoagulant which inhibits the endotoxin-mediated activation of Limulus coagulation system

Exposure of $\text{Limulus}$ amebocytes to bacterial endotoxins (lipopolysaccharides, LPS) results in the activation of the coagulation system, which consists of several protein components. During the separation of these components, a potent anticoagulant, named tentatively anti-LPS factor, which inhibits the endotoxin-mediated coagulation reaction, was found in both amebocytes from the hemolymphs of $\text{Tachypleus}\text{tridentatus}$ and $\text{Limulus}\text{polyphemus}$. The principle purified partially from $\text{Tachypleus}$ amebocyte lysate had a molecular weight less than 10,000, as judged with the ordinary gelfiltration experiment. It inhibited specifically the activation of factor B, which has recently been characterized to be a coagulation factor highly sensitive to LPS, but it did not inhibit the activities of the active factor B and the active clotting enzyme separated from the lysate. The inhibitory activity of anti-LPS factor disappeared almost completely by the treatments with pronase-P and subtilisin, suggesting its polypeptide-like substance, but it resisted to a boiling treatment. A possible site of the anticoagulant action on the $\text{Limulus}$ coagulation system was discussed.     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.     

The effects of thyroxine and methimazole treatment on the synthesis of prostacyclin (PGI2) in the rat

The effects of thyroxine (T4) and methimazole administration on plasma prostacyclin (PGI₂) levels $\text{in vivo}$ and on PGI₂ release by aortic rings incubated $\text{in vitro}$ were investigated in rats. Male rats were given single injection of T4 (200 μg/100 g body wt) ip every 24 h for either 3, 7 or 14 days for hyperthyroid rats. For hypothyroid rats, a group of rats were given methimazole (0.01 % in drinking water) for 14 days. PGI₂ concentrations were determined in plasma and also in the medium in which aortic rings were incubated. PGI₂ was measured as 6-keto-PGF1α by RIA. Plasma PGI₂ levels in T₄-treated groups were found to be significantly higher than those of control animals. Aortic rings obtained from rats given single injection of T₄ for 7 and 14 days showed significant increases in release of PGI₂ into the incubation medium. In contrast, rats given methimazole for 14 days showed a significant decrease in the production of PGI₂ by aortic rings without any significant changes in plasma levels. Direct addition of T₄ into the incubation medium did not cause any significant changes in PGI₂ release by aortic rings obtained from control rats.These results suggest the regulatory role of thyroid hormone in PGI₂ synthesis $\text{in vivo}$.     

Purification and characterization of an intracellular N-terminal exopeptidase from Streptococcus durans

An intracellular N-terminal exopeptidase isolated from cell extracts of Streptococcus durans has been purified 470-fold to homogeneity (specific activity of 12.0 μmol/min per mg). In the absence of thiol compounds, the purified aminopeptidase undergoes a slow oxidation with a 70% loss of activity, which can be restored by the addition of 2 mM β-mercaptoethanol. The purified aminopeptidase (M_r 300 000) preferred L-peptide and arylamide substrates with small nonpolar or basic side chains. SDS electrophoresis yielded a single protein band corresponding to a molecular weight of 49 400, suggesting that the native enzyme is a hexameric protein. The enzyme-catalyzed hydrolysis of $\text{L}\text{-}\text{alanyl}\text{-p-}\text{nitroanilide}$ exhibited a bell-shaped pH dependence for log V_max/K_m(pK₁ = 6.35; pK₂ = 8.50) while the log V_max versus pH profile showed only an acid limb (pK = 6.35). Methylene blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino acid analysis indicated that this photooxidative loss of activity corresponded to the modification of one histidine residue per enzyme monomer. N-Ethylmaleimide (100 mM) caused a 78% reduction in enzyme activity. Treatment of the enzyme with 1.0 mM hydrogen peroxide resulted in the oxidation of two cysteine residues per enzyme monomer and caused a 70% decrease in the catalytic activity.     

Diurnal variations in activity of four pyridoxal enzymes in rat liver during metabolic transition from high carbohydrate to high protein diet.

The diurnal variations in enzyme activities including tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT) and serine dehydratase (SDH) have been studied in rats trained to a 2 hour meal feeding schedule (″2+22″) during metabolic transition from 12.5 to 60% protein diets over a period of 21 days. Although the maximal TAT activity on the first day was slightly lower compared with other days, both TAT and ODC activities adapted rapidly to the increased dietary protein from the first day. The responses of TAT and ODC to the food were so rapid that the maximal value was observed only 4 hrs after the onset of feeding. After each feeding ODC activity decreased rapidly after 4 hours, while TAT activity declined only after 6 hours had elapsed. No clear diurnal rhythm was observed in either OAT or SDH, though OAT activity tended to decrease from the beginning of the dark period and to resume a slow adaptation after about four hours. In contrast to ODC and TAT both OAT and SDH required about 7 days to fully adapt to the high protein diet. The activities of the four enzymes were also compared after 4 groups of rats had been adapted to the ″2+22″ feeding of 12.5, 30 and 60% protein diets and to 60% diet, $\text{ad}$$\text{libitum}$, respectively. The enzyme activities were not directly proportional to the protein content of the diets although higher activity was observed on the high protein diets. The diurnal variations in both TAT and ODC were observed in all ″2+22″ groups although the timing of the peak values were slightly different from each other. The maximal activities of TAT were found at earlier times in 12.5 and 30% protein groups than in the 60% protein group. The peak time for ODC activity was found at a later time in the 12.5% protein group than in rats fed 30% and 60% protein. $\text{Ad}$$\text{libitum}$ rats fed 60% protein maintained relatively high levels of TAT activity compared to the rats on the schedule. However, the maximal activity of ODC on the 60% ″2+22″ protein diet $\text{ad}$$\text{libitum}$ was so low that a diurnal rhythm was not clearly evident.