Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits

Highly purified mitochondrial ribosomes (mitoribosomes) have been obtained from the yeast Candida utilis. Sedimentation analysis in sucrose gradients made in 5 mM MgCl₂, 1 mM Tris, pH 7.4 and 50 mM KCl clearly distinguishes mitoribosomes (72S) from cytoplasmic ribosomes (cytoribosomes) (78S). Mitoribosomes are completely dissociated into 50S and 36S subunits at 10^-4 M MgCl₂ whereas complete dissociation of cytoribosomes into 61S and 37S subunits occurs only at 10^-6 M MgCl₂ Electron microscopy of negatively stained mitoribosomes (72S peak) shows bipartite profiles, about 265 x 210 x 200 A Characteristic views are interpreted as frontal, dorsal, and lateral projections of the particles, the latter is observed in two enantiomorphic forms Mitoribosome 50S subunits display rounded profiles bearing a conspicuous knoblike projection, reminiscent of the large bacterial subunit. The 36S subunits show a variety of angular profiles. Mitoribosomal subunits are subject to artifactual dimerization at high Mg²⁺ concentration Under these conditions, a supplementary 80S peak arises. Electron microscopic observation of the 80S peak reveals closely paired particles of the 50S type Buoyant density determinations after glutaraldehyde fixation show a single peak at ρ = 1.48 for mitoribosomes and 1.53 for cytoribosomes In the presence of ethylenediaminetetraacetate (EDTA), two species of RNA, 21S and 16S, are obtained from mitoribosomes, while 25S and 17S RNA are obtained from cytoribosomes It is established that the small and large RNA species are derived from the 36S and 50S subunits, respectively, by extraction of the RNA from each subunit The G + C content of the RNA is lower for mitoribosomes (33%) than for cytoribosomes (50%). Incubation of C utilis mitochondria with leucine-¹⁴C results in the labeling of 72S mitoribosomes. The leucine-¹⁴C incorporation is inhibited by chloramphenicol and resistant to cycloheximide Puromycin strips the incorporated radioactivity from the 72S mitoribosomes, which is consistent with the view that leucine-¹⁴C is incorporated into nascent polypeptide chains at the level of mitoribosomes     

The sedimentation behaviour of ribonuclease-active and -inactive ribosomes from bacteria

1. The `30s' and `50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride–magnesium chloride solution, I 0·16, made 0–50mm with respect to Mg²⁺. 2. Differentiation of enzymic and physical breakdown at Mg²⁺ concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of `50s' into 40–43s components. This was combined with the release of a small amount of `5s' material which may be covalently bound soluble RNA. Other transformations of the `50s' into 34–37s components were observed in both ribonuclease-active and -inactive ribosomes at 1·0–2·5mm-Mg²⁺, and also with E. coli MRE600 when EDTA (0·2mm) was added to a solution in 0·16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg²⁺ concentration 0·25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs⁺/Mg²⁺ ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.     

Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid

Thin strips of frog ventricle were isolated and bathed for 15 min in a solution containing 140 mM KCl, 5 mM Na₂ATP, 3 mM EDTA, and 10 mM Tris buffer at pH 7.0. The muscle was then exposed to contracture solutions containing 140 mM KCl, 5 mM Na₂ATP, 1 mM MgCl₂, 10 mM Tris, 3 mM EGTA, and CaCl₂ in amounts to produce concentrations of free calcium from 10^-4.8 M to 10^-9 M. The muscles developed some tension at approximately 10^-8 M, and maximum tension was achieved in 10^-5 M Ca⁺⁺. They relaxed in Ca⁺⁺ concentrations less than 10^-8 M. The development of tension by the EDTA-treated muscles was normalized by comparison with twitch tension at a stimulation rate of 9 per min before exposure to EDTA. In 10^-5 M Ca⁺⁺ tension was always several times the twitch tension and was greater than the contracture tension of a frog ventricular strip in KCl low Na-Ringer. Tension equal to half-maximum was produced at approximately 10^-6.2 M Ca⁺⁺. Intracellular recording of membrane potential indicated that after EDTA treatment the resting potential of cells in Ringer solution with 10^-5 M Ca or less was between 5 and 20 mv. Contracture solutions did not produce tension without prior treatment with EDTA. The high permeability of the membrane produced by EDTA was reversed and the normal resting and action potentials restored in 1 mM Ca-Ringer. Similar studies of EDTA-treated rabbit right ventricular papillary muscle produced a similar tension vs. Ca⁺⁺ concentration relation, and the high permeability state reversed with exposure to normal Krebs solution.     

Effects of the Intracellular Ca Ion Concentration upon the Excitability of the Muscle Fiber Membrane of a Barnacle

The membrane excitability and contraction were examined in single barnacle muscle fibers with different internal Ca⁺⁺ concentrations by using buffer solutions made up with EGTA and Ca-gluconate in various proportions. During the passage of dc currents the membrane shows all-or-none spike potentials for internal Ca⁺⁺ concentrations below about 8 x 10^-8 M, oscillatory potential changes in the range between 8 x 10^-8 to 5 x 10^-7 M, but neither oscillatory nor spike potentials were seen for concentrations above 5 x 10^-7 M. All-or-none spike potentials were suppressed when the internal Mg⁺⁺ concentration exceeded 5 mM. The suppression threshold of the internal Ca⁺⁺ concentration for the Sr spike is much higher than that for the Ca spike. The threshold concentration of internal Ca⁺⁺ for contraction was about 8 x 10^-7 M.     

Requirement for extraction of polyribosomes from barley tissue

The isolation of barley (Hordeum vulgare L.) polyribosomes, showing minimal degradation effects of endogenous RNase, required a buffer adjusted to pH 8.0 and containing 0.40 m KCl in addition to common extraction components. The extracted polyribosomes were characterized in sucrose gradients by their conversion to monosomes when incubated with pancreatic RNase and by their dependence on adequate amounts of Mg²⁺ during extraction and analysis. Factors which contributed to polyribosome stability were evaluated by the relative sedimentation rates of aggregates in sucrose gradients. Tissue extraction at KCl concentrations less than 0.40 m and below pH 8.0 resulted in an appearance of larger amounts of ribosomes in the less dense region of the sucrose gradient after centrifugation. The addition of 10 mm dithiothreitol was partially effective in preventing the loss of higher polymerized states of polyribosomes at KCl concentrations below 0.40 m. Extractions conducted at KCl concentrations greater than 0.40 m and at pH 8.0 reduced the amount of ribosomes obtained from the tissue. The monosome portion of the polyribosomal profile was partially dissociated into subunits when the tissue was extracted in 0.60 m KCl. A similar effect on monosomes was obtained when polyribosomes were incubated with cycloheximide and 0.40 m KCl, a result not observed by use of a combination of 0.10 m KCl and the drug or 0.40 m KCl alone.     

Thermal Analysis of Bacterial Ribosomes

Ribosomal particles of E. coli were examined by using a heat leakage scanning calorimeter. Remarkable changes were observed in thermograms of 70S ribosomes and their subunits when the Mg²⁺ concentration was raised from 1 mm to 10 mm. It was suggested that ribosomal subunits exist in more than one conformation, and changes in their conformation might be the primary cause of the association-dissociation process of ribosomes. Comparisons of thermograms of RNase- and chymotrypsin-treated, as well as non-treated SOS and 30S subunits suggest that conformational changes in each subunit may be ascribed to changes in rRNA.     

Sarcoplasmic reticulum. IX. The permeability of sarcoplasmic reticulum membranes

Fragmented sarcoplasmic reticulum (FSR) membranes isolated from rabbit skeletal muscle are impermeable to inulin-¹⁴C (mol wt 5,000), and dextran-¹⁴C (mol wt 15,000–90,000) at pH 7.0–9.0, yielding an excluded space of 4–5 µl/mg microsomal protein. In the same pH range urea and sucrose readily penetrate the FSR membrane. EDTA or EGTA (1 mM) increased the permeability of microsomes to inulin-¹⁴C or dextran-¹⁴C at pH 8–9, parallel with the lowering of the FSR-bound Ca⁺⁺ content from initial levels of 20 nmoles/mg protein to 1–3 nmoles/mg protein. EGTA was as effective as EDTA, although causing little change in the Mg⁺⁺ content of FSR. The permeability increase caused by chelating agents results from the combined effects of high pH and cation depletion. As inulin began to penetrate the membrane there was an abrupt fall in the rate of Ca⁺⁺ uptake and a simultaneous rise in ATPase activity. At 40°C inulin penetration occurred at pH 7.0 with 1 mM EDTA and at pH 9.0 without EDTA, suggesting increased permeability of FSR membranes. This accords with the higher rate of Ca⁺⁺ release from FSR at temperatures over 30°C. The penetration of microsomal membranes by anions is markedly influenced by charge effects. At low ionic strength and alkaline pH acetate and Cl are partially excluded from microsomes when applied in concentrations not exceeding 1 mM, presumably due to the Donnan effect. Penetration of microsomal water space by acetate and Cl occurs at ionic strengths sufficiently high to minimize charge repulsions.     

Ca 2+ -specific removal of Z lines from rabbit skeletal muscle

Removal of rabbit psoas strips immediately after death and incubation in a saline solution containing 1 mM Ca²⁺ and 5 nM Mg²⁺ for 9 hr at 37°C and pH 7.1 causes complete Z-line removal but has no ultrastructurally detectable effect on other parts of the myofibril. Z lines remain ultrastructurally intact if 1 mM 1,2-bis-(2-dicarboxymethylaminoethoxy)-ethane (EGTA) is substituted for 1 mM Ca²⁺ and the other conditions remain unchanged. Z lines are broadened and amorphous but are still present after incubation for 9 hr at 37°C if 1 mM ethylenediaminetetraacetate (EDTA) is substituted for 1 mM Ca²⁺ and 5 mM Mg²⁺ in the saline solution. A protein fraction that causes Z-line removal from myofibrils in the presence of Ca²⁺ at pH 7.0 can be isolated by extraction of ground muscle with 4 mM EDTA at pH 7.0–7.6 followed by isoelectric precipitation and fractionation between 0 and 40% ammonium sulfate saturation. Z-line removal by this protein fraction requires Ca²⁺ levels higher than 0.1 mM, but Z lines are removed without causing any other ultrastructurally detectable degradation of the myofibril. This is the first report of a protein endogenous to muscle that is able to catalyze degradation of the myofibril. The very low level of unbound Ca²⁺ in muscle cells in vivo may regulate activity of this protein fraction, or alternatively, this protein fraction may be localized in lysosomes.     

Studies on streaming. I. Inhibition of protoplasmic streaming and cytokinesis of Chaos chaos by adenosine triphosphate and reversal by magnesium and calcium ions

In Chaos chaos streaming, motility, and cytokinesis were inhibited nearly 100% for several hours by 2.5–5 mM sodium adenosine triphosphate (ATP)¹ added to culture fluid. All three effects were completely prevented by the addition of equimolar Mg⁺⁺ or Ca⁺⁺ ions but not Na⁺ to the ATP/culture fluid solution. The effects of ATP were not reproduced by EDTA, EGTA, colchicine, or AMP. Sodium pyrophosphate produced about 50% inhibition at 5 mM. Studies with ¹⁴C-ATP showed that 5 x 10^-5 to 5 x 10^-4 mmole of ATP was firmly associated with each milliliter of packed cells after an hour''s incubation at 24°C. Labeling studies also showed that prevention of the ATP effects by Mg⁺⁺ ions was not due to a decrease in the amount of ATP associated with the cells.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

CYTOCHEMICAL STUDY ON THE PANCREAS OF THE GUINEA PIG : VII. Effects of Spermine on Ribosomes

Pancreatic ribosomes (guinea pig) aggregate and lose upon treatment with polyamines, particularly spermine, their bound secretory enzymes. Spermine, at 0.5 mM, for example, causes the release of about 85 per cent of the chymotrypsinogen and RNase, and from 85 to 100 per cent of the ribosomal amylase. At the same time, the particles lose about 10 per cent of their RNA, 7 to 24 per cent of their total protein, and from 75 to 100 per cent of their Mg⁺⁺. Observations with the electron microscope confirm the heavy agglutinating of the ribosomes but otherwise show little change in the structure of the particles. Using radioactive spermine it was found that, concomitant with the loss of bound enzymes and Mg⁺⁺ from the ribosomes, spermine became bound to the particle. The extent of binding ranged from 0.29 to 1.49 µmoles per 10µmoles RNA-P. The bound radioactive spermine can be removed by subsequent treatment of the ribosomes with GTP, ATP, or P-P, which treatment also removes most of the RNA of the particles, leaving behind ribosomes with a much lower RNA/protein ratio. From this evidence it was inferred that spermine, in releasing the Mg⁺⁺ of the particle, becomes salt-linked to the free phosphate hydroxyl groups of the RNA. Freshly isolated pancreatic and hepatic ribosomes contain very little spermine, about 0.1 to 0.2 µmoles polyamine/10 µmoles RNA-P. The results are discussed in terms of the linkages between the structural protein, the bound secretory enzymes, and the RNA of the ribosomes.     

Increased Activity of Chromatin-bound Ribonucleic Acid Polymerase from Soybean Hypocotyl with Spermidine and High Ionic Strength

Optimal activity of chromatin-bound RNA polymerase from soybeans is obtained with 1 mm Mn²⁻, but only when high ionic strength or polyamines are included in the medium. Such inclusion does not increase the Mg²⁺ activation of the polymerase, but it does lower the concentration needed for optimum activity from 10 mm to 1 mm. Mg²⁻ activation is inhibited by added Mn²⁺, and the inhibition is relieved by high ionic strength or spermidine. The RNA polymerase with either cation is almost entirely polymerase I at low and high ionic strength as evidenced by insensitivity to α-amanitin. Treatment of soybean seedlings with 2,4-dichlorophenoxyacetic acid does not change these characteristics; although the activity rises 3- to 4-fold.     

Dissociation of N(2) Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea

The dissociation of N₂ gas-induced monomeric ribosomes from the pea root was studied by varying the concentration of KCl (or NH₄Cl) and MgCl₂ in the presence of dithiothreitol. These monoribosomes were shown to dissociate completely into subunits at 0.5m KCl or NH₄Cl in the presence of 5 mm MgCl₂. The 40S subunits were more susceptible to structural change in KCl than were the 60S subunits. On the other hand, the 60S subunits appeared to be more labile to NH₄Cl.     

Nature of the Schwann cell electrical potential. Effects of the external ionic concentrations and a cardiac glycoside

The effects on the Schwann cell electrical potential of external ionic concentrations and of K-strophanthoside were investigated. Increasing (K)_o depolarized the cell. The potential is related to the logarithm of (K)_o in a quasi-linear fashion. The linear portion of the curve has a slope of 45 mv/ten-fold change in (K)_o. Diminutions of (Na)_o and (Cl)_o produced only small variations in the potential. Calcium and magnesium can be replaced by 44 mM calcium without altering the potential. Increase of (Ca)_o to 88 mM produced about 10 mv hyperpolarization. The cell was hyperpolarized by 11 mv and 4 mv within 1 min after applying K-strophanthoside at concentrations of 10^-3 and 10^-5 M, respectively. No variations of cellular potassium, sodium, or chloride were observed 3 min after applying the glycoside. The hyperpolarization caused by 10^-3 M K-strophanthoside was not observed when (K)_o was diminished to 1 or 0.1 mM or was increased to 30 mM. At a (K)_o of 30 mM, 10^-2 M strophanthoside was required to produce the hyperpolarizing effect. In high calcium, the cell was further hyperpolarized by the glycoside. The initial hyperpolarization caused by the glycoside was followed by a gradual depolarization and a decrease of the cellular potassium concentration. The results indicate that the Schwann cell potential of about -40 mv is due to ionic diffusion, mainly of potassium, and to a cardiac glycoside-sensitive ion transport process.     

Loosening and unfolding of E. coli 50 S ribosomal subunits: Dependence on magnesium content and temperature

Reversible change of 50 S ribosomal subunits to 40 S particles takes place in cold buffered 0.5 M NH₄Cl solutions either containing Mg⁺⁺ (up to 0.1 M), or free from Mg⁺⁺ and even supplemented with EDTA (1 mM). The 40 S particles were stable only within a definite temperature range. Heating of the samples caused completely irreversible unflding of the 40 S particles. This melting appeared to be co-operative and took place within a very narrow range of temperature, which for samples containing Mg⁺⁺ was a linear function of the log of Mg⁺⁺ concentration.The results suggest that two types of bonds maintained the compact structure of the ribosomal subunits: ionic bonds involving Mg⁺⁺ and heat-labile weak interactions between ribosomal components.     

Effects of Sodium Azide on Sodium Fluxes in Frog Striated Muscle

Unidirectional Na fluxes from frog''s striated muscle were measured in the presence of 0 to 5 mM sodium azide. With azide concentrations of 2 and 5 mM the Na efflux was markedly stimulated; the Na efflux with 5 mM azide was about 300 per cent greater than normal. A similar increase was present when all but the 5.0 mM sodium added with azide was replaced by choline. 10^-5 M strophanthidin abolished the azide effect on Na²⁴ efflux. Concentrations of azide of 1.0 mM or less had no effect on Na efflux. The Na influx, on the other hand, was only increased by 41 per cent in the presence of 5 mM NaN₃. From these findings it is concluded that the active transport of Na is stimulated by the higher concentrations of azide. The hypothesis is advanced that the active transport of Na is controlled by the transmembrane potential and that the stimulation of Na efflux is produced as a consequence of the membrane depolarization caused by the azide.     

The Dual Effect of Lithium Ions on Sodium Efflux in Skeletal Muscle

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3°C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer''s solution containing 60 mM LiCl and 50 mM NaCl at 20°C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10^-5 M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer''s solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10^-5 M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer''s solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.     

Methods of cell disintegration and the sedimentation patterns of ribosomes inEscherichia coli

A number of methods for the disintegration ofEscherichia coli cells was followed to investigate their suitability for the isolation of ribosomes. The decrease of viability and the conditions for fine disruption of the cells for all methods were established. The sedimentation coefficients of ribosomes and their sizedistribution in the cell-free extracts in dependence upon the method employed and the concentration of Mg⁺⁺ ions were determined by analytical ultracentrifugation. The sedimentation coefficients and the sizedistribution of ribosomes did not differ in most methods and the results obtained were basically in agreement with the data given by other authors. The method of grinding with alumina seemed to be too vigorous and the results obtained were less reproducible. Ribosomes from extracts prepared by osmotical shocking of lysozyme spheroplasts showed ultracentrifugation patterns differing from those found with other methods: In extracts with low (0.00025m) and high (0.01m) concentration of Mg⁺⁺ almost the whole range of ribosomes was found whereas in the buffer with 0.001m Mg⁺⁺ only 30 and 50s ribosomes were present. alumina Particles with a sedimentation constant higher than 100s (polysomes) were not found in any of our extracts. We attempted to discuss our results and compare them with the data given in other reports. The method of osmotical lysis of spheroplast seems to give results most closely related to thein vivo situation in the cell and enables us to study the functional relationship of ribosomes to other cell components namely the cytoplasmic membrane.     

The influence of ionic strength on potassium contractures and calcium movements in frog muscle

Frog toe muscles were bathed in isotonic, sodium-free Tris chloride, methanesulfonate, or sulfate solutions containing sucrose or mannitol and varying in ionic strength from 0.006 to 0.291. By decreasing the ionic strength the curve relating the peak tension of the K contractures to the log [K] was reversibly shifted to lower [K]. Increasing the [Ca] from 1 to 4 mM almost abolished this effect. The resting uptake of ⁴⁵Ca was increased more than two times by decreasing the ionic strength from 0.125 to 0.039. It was not increased significantly by raising [Ca] from 1 to 4 mM at low or normal ionic strength. The additional uptake of ⁴⁵Ca during contractures provoked by 120 mM K was not significantly different at the two levels of ionic strength. The rate of emergence of ⁴⁵Ca from muscles loaded with ⁴⁵Ca at reduced ionic strength, was decreased. The effects of low ionic strength are discussed in terms of changes in the potential difference across a membrane with fixed negative charges on the outer surface.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).