Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.     

Solubilization of adenylate cyclase from Ceratitis capitata neural membranes and incorporation into liposomes

Solubilization of the adenylate cyclase from neural membranes of the dipterous Ceratitis capitata, by using several detergents, and regulatory characteristics of the solubilized enzyme were examined. Triton X-100 is the most effective detergent in solubilizing this enzyme activity. The adenylate cyclase in Triton X-100-solubilized preparations (105,000 g supernatant) does not respond to either guanine nucleotides or fluoride and it apparently seems to be devoid of a functional regulatory component. When this preparation is centrifuged again at 300,000 g for 30 min no enzyme activity is detectable in the supernatant, however only 8% of total activity is recovered in the pellet. The activation pattern for the enzyme in the 300,000 g pellet is similar to that observed for the enzyme in the 105,000 g supernatant. Incorporation of solubilized enzyme into dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC) or cholesterol-enriched DOPC liposomes increases the 300,000 g pellet adenylate cyclase activity in a similar extension; thus, this increase in enzyme activity appears to be independent not only on the phospholipid composition but also on the liposome fluidity.     

Biosynthesis and genetic control of isovitexin 7-O-xyloside in the petals of melandrium album

An enzyme was detected in petal extracts of Melandrium album which catalysed the transfer of the xylose moiety of UDP-xylose to the 7-hydroxyl group of isovitexin. Genetical analysis revealed that the presence of the dominant allele g^x was necessary for enzymic activity. This activity was independent of the residual genetic background. Xylosyltransferase activity is also present in extracts of g^Gg^x plants, in which the product of the enzyme is not detectable. Maximal activity was found between pH 7·0 and 7·5; MnCl₂ inhibited this transfer. The enzyme had an ‘apparent K_m' value of 1·0 mM for UDP-xylose and of O·04 mM for isovitexin.     

ATPase in ripening strawberries

ATPase was found in 1000g, 13 000g and 80 00Og fractions from strawberry fruits. The optima pH for ATPase was the same (i.e. 6) for the 3 fractions, which also showed similar substrate specificity. However, the enzyme associated with the 80 000 g fraction showed the highest affinity for ATP and the maximum V_max/K_m value. As the fruit ripened, from the green to dark-red stage, ATPase activity in the 80 000 g fraction increased more than three times. The ATP content of the fruit pulp, which was high at the green stage, decreased as the fruit matured and ripened. Na⁺ and K⁺ slightly stimulated enzyme activity associated with the 1000 g,80 000 g and soluble fractions, whereas, Ca²⁺ and Mg²⁺ inhibited the enzyme activity in all fractions. However, the extent of inhibition due to divalent cations lessened as the fruit ripened.     

Bovine adrenocortical adenylate cyclase: Some properties of the solubilized,fluoride-activated enzyme

The activity of bovine adrenocortical plasma membrane adenylate cyclase can be maintained at 4°C in the presence of NaF. The half-life of the fluoride-stabilized enzyme is approximately 7 days. Maximal activation by fluoride requires approximately 20 min at 0°C and the level of activity attained is dependent on fluoride concentration. The enzyme from freshly harvested membranes can also be stimulated by ACTH_{1 – 24} and Gpp(NH)p and the stimulatory effects of these two activators are additive. Prolonged exposure to either NaF or Gpp(NH)p precludes hormone activation. Optimal concentration for Gpp(NH)p activation is 10^?4–10^?5M. Treatment of the enzyme with a Tris-HCl buffer containing Lubrol-PX (1%), NaF, dithiothreitol, and MgSO₄ followed by sonication affords a preparation that does not sediment at 100 000 g in 1 hr. This material has a low specific activity; however, removal of the detergent on DEAE cellulose restores specific activity to its original level. A significant improvement in specific activity is observed following dialysis or ultrafiltration of the detergent-free 100 000-g supernatant. At this stage the enzyme can be lyophilized and stored at ?70°C without loss of activity. The enzyme in the detergent-free 100 000-g supernatant behaves as a single peak that is included in Sepharose 6B. Comparison of the elution profile of the enzyme with profiles produced by a standard set of proteins suggests a molecular weight of 1 × 10⁶. Hydrophobic chromatography of the detergent-free 100 000-g supernatant on n-hexyl Sepharose 4B results in a fivefold enhancement of specific activity.     

Formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774: isolation and spectroscopic characterization of the active sites (heme, iron-sulfur centers and molybdenum)

An air-stable formate dehydrogenase, an enzyme that catalyzes the oxidation of formate to CO₂, was purified from a sulfate-reducing organism, Desulfovibrio desulfuricans ATCC 27774. The enzyme has a molecular mass of approximately 150?kDa (three different subunits: 88, 29 and 16?kDa) and contains three types of redox-active centers: four c-type hemes, nonheme iron arranged as two [4Fe-4S]^2+/1+ centers and a molybdenum-pterin site. Selenium was also chemically detected. The enzyme specific activity is 78 units per mg of protein. Mo(V) EPR signals were observed in the native, reduced and formate-reacted states. EPR signals related to the presence of multiple low-spin hemes were also observed in the oxidized state. Upon reduction, an examination of the EPR data under appropriate conditions distinguishes two types of iron-sulfur centers, an [Fe-S] center I (g _max=2.050, g _med=1.947, g _min=1.896) and an [Fe-S] center II (g _max=2.071, g _med=1.926, g _min=1.865). Mössbauer spectroscopy confirmed the presence of four hemes in the low-spin state. The presence of two [4Fe-4S]^2+/1+ centers was confirmed, one of these displaying very small hyperfine coupling constants in the +1 oxidation state. The midpoint redox potentials of the enzyme metal centers were also estimated.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

Release of glucose-6-phosphate dehydrogenase from cortex of Spisula eggs at fertilization and its recombination after meiosis

Changes in subcellular distributions of glucose-6-phosphate dehydrogenase (G6PDH) were observed after fertilization or artificial (KCl) activation of Spisula eggs. Though the total activity of G6PDH did not change during early stages, that in the 100,000g supernatant fraction increased after fertilization, attained a maximum at the first meiotic metaphase, and then decreased. This change of activity in the supernatant was accompanied by a mirror-image change of activity in the pellet. Most of the G6PDH was localized in the 3000g pellet fraction; furthermore, the activity in isolated cortices showed fluctuations during meiosis similar to that of the 3000g pellet fraction. Conditions for the release and binding of the NADP-specific G6PDH from the pellet fraction were investigated in vitro. NADP⁺ or NADPH can induce release of G6PDH, although NADPH is three to four times more efficient than NADP⁺. NAD⁺ does not affect release. High concentrations of salts (ionic strength >0.3) caused complete G6PDH release from the pellet. Although raising the pH alone showed only a slight releasing effect, increase of pH to pH 7 or above considerably augmented release due to NADP⁺ or NADPH. The release of G6PDH from the pellet fraction was shown to be reversible. These results suggest that the reversible association of G6PDH with particulate components of the cytoplasm may play an important role in regulation of G6PDH activity in marine eggs and that the cortex is one of the sites which may be involved in such regulation. The mechanism of recombination of G6PDH with its sites remains to be elucidated.     

Genetic Analysis of Natural Populations of DROSOPHILA MELANOGASTER in Japan. III. Genetic Variability of Inducing Factors of Amylase and Fitness

"Inducibility" of amylase in Drosophila melanogaster was defined and investigated in a natural population from Japan. Inducibility represents the effects of factors remote from the structural gene that control the amount of enzyme produced. Inducibility of an isogenic line is measured as the ratio of the enzyme's specific activity in two different inducing environments. There was considerable genetic variability with respect to inducibility of amylase in 44 isogenic lines derived from a natural population of D. melanogaster . Net fitness and its components in these isogenic lines were also measured. The results indicated that, although the inducibility of the enzyme was positively correlated with the net fitness (r_g = 0.63 ± 0.2), the enzyme activities in the normal medium were not (r_g = 0.12 ± 0.37). The analysis of the data shows that the differences in inducing factors are mainly responsible for the differences in the fitness of lines and are the genetic materials for the adaptive evolution of organisms.     

Calorie restriction influences key metabolic enzyme activities and markers of oxidative damage in distinct mouse liver mitochondrial sub-populations

Aims

The purpose of the study was to establish if enzyme activities from key metabolic pathways and levels of markers of oxidative damage to proteins and lipids differed between distinct liver mitochondrial sub-populations, and which specific sub-populations contributed to these differences.

Main methods

Male C57BL/6J mice were fed non-purified diet for one month then separated into two groups, control and calorie-restricted (CR). The two groups were fed semi-purified diet (AIN93G), with the CR group receiving 40% less calories than controls. After two months, enzyme activities and markers of oxidative damage in mitochondria were determined.

Key findings

In all mitochondrial sub-populations, enzyme activities and markers of oxidative damage, from control and CR groups, showed a pattern of M1 > M3 > M10. Higher acyl-CoA dehydrogenase (β-oxidation) and β-hydroxybutyrate dehydrogenase (ketogenesis) activities and lower carbonyl and TBARS levels were observed in M1 and M3 fractions from CR mice. ETC enzyme activities did not show a consistent pattern. In the Krebs cycle, citrate synthase and aconitase activities decreased while succinate dehydrogenase and malate dehydrogenase activities increased in the M1 mitochondria from the CR versus control mice.

Significance

CR does not produce uniform changes in enzyme activities or markers of oxidative damage in mitochondrial sub-populations, with changes occurring primarily in the heavy mitochondrial populations. Centrifugation at 10,000 g to isolate mitochondria likely dilutes the mitochondrial populations which show the greatest response to CR. Use of lower centrifugal force (3000 g or lower) may be beneficial for some studies.     

Sub-mitochondrial location of Ruthenium Red-sensitive calcium-ion transport and evidence for its enrichment in a specific population of rat liver mitochondria

1. Seven fractions sedimenting at between 3000 and 120000g-min were prepared from a rat liver homogenate by differential centrifugation in buffered iso-osmotic sucrose. The following measurements were carried out on each of these fractions: Ruthenium Red-sensitive Ca²⁺ transport in the absence and in the presence of P_i as well as in the presence of N-ethylmaleimide to prevent P_i cycling, succinate-supported respiration in the absence and in the presence of ADP, the ΔE and −59 ΔpH components of the protonmotive force, cytochrome oxidase, uncoupler-stimulated adenosine triphosphatase, α-glycerophosphate dehydrogenase, P_i content and the effect on the `resting' rate of respiration of repeated additions of a fixed Ca²⁺ concentration. 2. Ca²⁺ transport either in the presence or in the absence of added P_i and in the presence of N-ethylmaleimide exhibits significantly higher rates in the fraction sedimenting at 8000g-min. By contrast, respiration in the presence or in the absence of added ADP and the values for ΔE and −59 ΔpH were similar in those fractions sedimenting between 4000 and 20000g-min, indicating that the driving force for Ca²⁺ transport was similar in each of these fractions. 3. Experiments designed to determine the capacity of the individual fractions for Ca²⁺, as measured by the effect of repeated additions of Ca²⁺ on the resting rate of respiration, showed that fraction 2, i.e. that sedimenting at 8000g-min, also exhibited the greatest tolerance towards the uncoupling action of the ion. 4. Of the three enzyme activity profiles, only that of α-glycerophosphate dehydrogenase was similar to that of Ca²⁺ transport. Because previous workers have assigned this enzyme to loci in the inner peripheral membrane [Werner & Neupert (1972) Eur. J. Biochem. 25, 379–396], it is concluded that the Ruthenium Red-sensitive Ca²⁺- transport system also is located in this domain of the inner membrane. The relation of these findings to the mechanisms of mitochondrial Ca²⁺ transport and the biogenesis of mitochondria is discussed.     

EPR studies of the oxygen-evolving enzyme of Photosystem II

The light-induced EPR multiline signal is studied in O₂-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S₂ state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q^?_AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S₂. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S₂ signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.     

FINE STRUCTURE AND PIGMENT CONVERSION IN ISOLATED ETIOLATED PROPLASTIDS

Proplastids containing a prolamellar body were isolated from leaves of etiolated bean plants. The isolation methods do not necessarily lead to destruction of their submicroscopic structure and most of the isolated proplastids show well preserved outer membranes, lamellar strands, and the prolamellar body. Morphological intactness of the proplastids varies; certain leaf fractions contain single prolamellar bodies as well as proplastids. Since pellets after centrifugation between 350 g and 1000 to 3000 g contain intact proplastids and, as was shown by quantitative experiments, the same fractions show photoconversion of protochlorophyll to chlorophyll, it is supposed that the isolated particles probably retain many of the properties which are characteristic of them in situ. Isolated proplastids may thus be a valuable tool in investigations on the development of the photosynthetic apparatus.     

An elastase-like enzyme in the oocytes of Arbacia Punctulata.

An elastase-like enzyme was demonstrated in the unfertilized eggs of Arbacia punctulata. The enzyme was discovered in the fertilization product of A. punctulata, but was also found to be present in the seawater surrounding unfertilized eggs. Isolation of the elastase-like enzyme was accomplished by preparation of a 100,000g supernatant from a homogenization of unfertilized eggs. Its presence and specificity were determined by assay using a synthetic peptide substrate. The elastase-like activity was nondialyzable, heat labile, and unstable at pH 4. The enzyme was inhibited by antipain, elastatinal, and DFP, but not by leupeptin or soybean trypsin inhibitor. Eggs were fertilized and developed normally in the presence of 1.0 mM elastatinal. Trypsin-like and chymotrypsin-like enzymes were also found in the seawater surrounding fertilized eggs. The trypsin-like enzyme was isolated from this source and characterized by inhibitor profile.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

Enzyme supplementation of wheat-based diets for broilers: 1. Effect on growth performance and intestinal viscosity

The influence of enzyme supplementation on performance and intestinal viscosity of male broiler chickens fed with diets containing high amount of wheat was examined in three experiments. In the first experiment, addition with an enzyme preparation including different cell wall degrading enzymes to diets containing 63 g kg⁻¹ and 72 g kg⁻¹ of wheat improved (P<0.05) feed conversion efficiency in the 72 g kg⁻¹ wheat diet. In addition, intestinal viscosity of chickens fed with the 72 g kg⁻¹ wheat diet was reduced (P<0.05). Weight gain and feed intake were not influenced by enzyme addition. In Experiments 2 and 3, the inclusion level of wheat in the diets was more than 80 g kg⁻¹ and four different enzyme preparations were used (two xylanase preparations, two mixed preparations). Overall, a significant effect on performance and intestinal viscosity of chickens was obtained as a result of enzyme supplementation in both experiments. In the first 21 days, improvements (P<0.05) in weight gain and feed conversion efficiency were found to be on average 5% and 6% in Experiment 2 and 7% and 8% in Experiment 3, respectively. When weight gain and feed conversion efficiency were examined on a weekly basis it was shown that the significant response of enzyme addition was confined to the first 4 weeks. However, the effect of enzyme supplementation was still significant in the whole period from 21–42 days. Feed intake was not influenced by enzyme addition. The viscosity of intestinal content in both the jejunum and ileum was in general reduced (P<0.05) with enzyme supplementation, the xylanase preparations proving to be the most efficient. It was concluded that enzyme supplementation of wheat-based diets resulted in improved performance of broiler chickens, which was related to a concomitant reduction in intestinal viscosity. However, the response of enzyme supplementation was most pronounced in diets with a wheat content higher than 80 g kg⁻¹.     

Mn(III)-containing acid phosphatase: Properties of Fe(III)-substituted enzyme and function of Mn(III) and Fe(III) in plant and mammalian acid phosphatases

The function of Mn(III) in plant acid phosphatase has been investigated by a metal-substitution study, and some properties of the Fe(III)-substituted enzyme were compared with those of the native Mn(III) enzyme and mammalian Fe(III)-containing acid phosphatases. ¹⁹F nuclear magnetic resonance (NMR) and proton relaxation rate measurements showed that inhibitors such as F⁻ and nitrilotriacetic acid interact with paramagnetic Mn(III) active site. The ³¹P-NMR signal of the enzyme-phosphate complex was also broadened by the paramagnetic effect of Mn(III). In the metal-substitution experiments of the Mn(III)-acid phosphatase with Fe(III), Zn(II) and Cu(II), only the iron gave satisfactory substitution. The Fe(III)-substituted plant acid phosphatase exhibited an absorption maximum at 525 nm (ε = 3000), typical high spin ferric ESR signal at g = 4.39, and lower pH optimum (pH 4.8) than the native Mn(III)-enzyme (pH 5.8). The phosphatase activity of the Fe(III)-substituted enzyme was reduced to about 53% of that of the native enzyme. The substrate specificities of both metallophosphatases were remarkably similar, but different from that of the Fe(III)-containing uteroferrin. The present results indicate that Mn(III) and Fe(IIII) in the acid phosphatase play an important role on effective binding of phosphate and acceleration of hydrolysis of phosphomonoesters at pH 4–6.     

The carbohydrate-binding module of Fragaria × ananassa expansin 2 (CBM-FaExp2) binds to cell wall polysaccharides and decreases cell wall enzyme activities “in vitro”

A putative carbohydrate binding module (CBM) from strawberry (Fragaria × ananassa Duch.) expansin 2 (CBM-FaExp2) was cloned and the encoding protein was over-expressed in Escherichia coli and purified in order to evaluate its capacity to bind different cell wall polysaccharides “in vitro”. The protein CBM-FaExp2 bound to microcrystalline cellulose, xylan and pectin with different affinities (K_ad = 33.6 ± 0.44 mL g^?1, K_ad = 11.37 ± 0.87 mL g^?1, K_ad = 10.4 ± 0.19 mL g^?1, respectively). According to “in vitro” enzyme assays, this CBM is able to decrease the activity of cell wall degrading enzymes such as polygalacturonase, endo-glucanase, pectinase and xylanase, probably because the binding of CBM-FaExp2 to the different substrates interferes with enzyme activity. The results suggest that expansins would bind not only cellulose but also a wide range of cell wall polymers.     

Studies of the copper and heme cofactors of pseudomonad L-tryptophan-2,3-dioxygenase by electron paramagnetic resonance spectroscopy

l-Tryptophan-2,3-dioxygenase, (EC 1.13.1.12) purified from Pseudomonas acidovorans, is inactivated on aerobic aging or on treatment with K₃Fe(CN)₆, but regains activity in the presence of reducing agents such as sodium ascorbate. Examination of oxidized, inactive enzyme by electron paramagnetic resonance (epr) spectroscopy has revealed the presence of high spin ferriheme (g = 6.2) and of Cu(II) (g_⊥ = 2.065, g_∥ = 2.265) in the enzyme.The epr signal of Cu(II) in inactive tryptophan oxygenase is attenuated on the addition of ascorbate, whereas the high spin ferriheme signal is unaffected, indicating that the site of action of reducing agents in activating the enzyme is the enzymic copper. Quantitation of the Cu(II) signal in inactive tryptophan oxygenase by double integration accounts for 45% of the total copper.Addition of l-tryptophan to either inactive or active enzyme produces a decrease of 44 ± 5% of the epr signal of high spin ferriheme and the emergence of the epr signal of a low spin ferriheme (g_{1, 2, 3} = 2.66, 2.20, 1.81). Disappearance of the high spin ferriheme is hyperbolic (Hill coefficient, n = 1.02) with respect to l-tryptophan concentration, while the appearance of the low spin ferriheme is sigmoidal (Hill coefficient, n = 1.33) with respect to l-tryptophan concentration. The characteristics of the epr signal of this low spin ferriheme are intermediate between those of the signals of the hydroxides of hemoglobin and myoglobin and those in which two histidines are ligated to the ferriheme of hemoglobin. This may be the first example of the observation by epr of an allosteric parameter of an enzyme.     

Studies on guanine deaminase and its inhibitors in rat tissue

1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.