Conversion of thyroxine to 3,3',5'-triiodothyronine (reverse-T3) by a soluble enzyme system of rat liver

Infrared spectra of N₂O in a variety of solvents and in the brain of a dog under typical conditions of halothane-N₂O anesthesia have been determined. The appearance or disappearance of N₂O in the brain was readily followed as N₂O was administered or withdrawn. The sites in brain were of two major types; one, with ν₃ = 2229.8 ± 0.4 cm^?1 and $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 13.0 ± 0.6}\text{cm}{}^{\mathrm{?1}}$, is rather like the polar site in water and the other, with ν₃ = 2216.8 ± 0.8 cm^?1 and $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.6 ± 1.0}\text{cm}{}^{\mathrm{?1}}$, is non-polar and is probably associated with membrane lipid. The significant variations in the antisymmetric stretch (ν₃) of N₂O as the polarity and other properties of the medium (solvent) vary make possible the characterization of $\text{in tissue}$ sites occupied by this anesthetic.     

Spatial requirements for insulin-sensitive sugar transport in rat adipocytes

(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (K_i = 9.05 ± 0.66 mM) is lost by N-acetylation. $\text{N-}\text{Acetyl-}\text{d}\text{-glucosamine}$ shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly $\text{2,3-}\text{di}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 42.1 ± 7.5 mM) has lower affinity than $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The K_i for $\text{3-O-}\text{propyl-}\text{d}\text{-glucose}$ is 11.26 ± 2.12 mM. $\text{6-O-}\text{Methyl-}\text{d}\text{-galactose}$ (K_i = 87.2 ± 17.9 mM) and $\text{6-O-}\text{propyl-}\text{d}\text{-glucose}$ (K_i = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in $\text{6-O-}\text{pentyl-}\text{d}\text{-galactose}$ (K_i = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells $\text{4,6-O-}\text{ethylidene-}\text{d}\text{-glucose}$ (K_i = 6.11 ± 0.5 mM) and maltose (K_i = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar K_i values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system.     

Dual action of ionophore A23187 on intact chloroplasts

1. Ionophore A23187 induces uncoupling of potassium ferricyanide-dependent O₂ evolution by envelope-free chloroplasts and oxaloacetate-dependent O₂ evolution by intact chloroplasts. The half maximal concentration ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for stimulation of oxygen evolution in both cases is approximately 4 μM · 100 μg chlorophyll · ml^?1.2. Ionophore A23187 also induces inhibition of CO₂ and 3-phosphoglycerate-dependent O₂ evolution by intact chloroplasts in the presence of 3 mM MgCl₂. The half maximal concentrations ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for inhibition of O₂ evolution are 3 μM and 5 μM respectively · 100 μg^?1 chlorophyll · ml^?1.3. A very high concentration of ionophore A23187 (10 μM · 20 μg^?1 chlorophyll · ml^?1) plus 0.1 mM EDTA lowers the fluorescence yield of intact chloroplasts suspended in a cation-free medium in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating loss of divalent cation from the diffuse double layers of the thylakoid membranes.4. These results are discussed in relation to ionophore A23187-induced divalent cation/proton exchange at both the thylakoid and the envelope membranes of intact chloroplasts.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

Regulation of activation of ribulose bisphosphate carboxylase from Pseudomonasoxalaticus

6-phosphogluconate, potentiated activation of ribulose bisphosphate carboxylase from $\text{Pseudomonas}\text{oxalaticus}$ whereas fructose-1,6-bisphosphate inhibited activation and fructose-6-phosphate had no effect. The presence of 1 mM 6-phosphogluconate during activation reduced the K_act for Mg²⁺ from 1.4 mM to approximately 0.2 mM. In the absence of 6-phosphogluconate, the enzyme responded sigmoidally to increasing CO₂ (Hill coefficient, h, of 1.8), with a concentration causing half maximal activation, Act_0.5, of 15 mM NaHCO₃. In the presence of 1 mM 6-phosphogluconate h was reduced to 1.1 and an Act_0.5 value of 5 mM NaHCO₃ was obtained. 6-phosphogluconate appeared to saturate at or below 20 μmM.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

The presence and binding characteristics of calmodulin in microsomal preparations enriched in sarcoplasmic reticulum from rabbit skeletal muscle

ATP-dependent oxalate facilitated calcium transport in sarcoplasmic reticulum (SR) preparations obtained from rabbit vastus lateralis muscle (fast skeletal muscle; F_sr) and soleus (slow skeletal muscle; S_sr) was determined. Addition of exogenous calmodulin did not stimulate calcium transport in either F_sr or S_sr preparations. F_sr and S_sr previously washed in 1 mM EGTA demonstrated a reduced capacity to transport Ca²⁺; the exogenous addition of calmodulin (0.24 μM) under these conditions, did not restore uptake activity but significantly decreased the steady-state level of Ca²⁺ uptake. Extracts of skeletal SR prepared by treatment with 0.2 mM EDTA and boiling produced significantly more stimulation of red cell Ca²⁺ATPase activity than extracts prepared by boiling alone. This stimulation of red cell Ca²⁺-ATPase was inhibited to a significant extent by $\text{48}\text{80}$, a known anti-calmodulin agent. Radioimmunoassay revealed that extracts prepared by boiling or EDTA-treatment followed by boiling contained considerable amounts of calmodulin. Washing with 1 mM EGTA, though, did not release any calmodulin from SR. These studies reveal that calmodulin is present in both F_sr and S_sr and can only be removed by harsh treatments. The role of calmodulin in skeletal muscle Ca²⁺-transport remains to be determined.     

Preparation of uniform haemoglobin free human erythrocyte ghosts in isotonic solution

A method is described for the preparation of haemoglobin free human erythrocyte ghosts in isotonic solutions using dielectric breakdown technique. In this single haemolytic procedure, almost complete removal of haemoglobin (? 0.1%) was achieved by subjecting the erythrocytes suspended in phosphate buffered, isotonic KCl solution at 0°C to three consecutive electrical field pulses of 16 kV/cm in the presence of 10 mM EDTA; EDTA was used to prevent electrical haemolysis. Haemolysis is induced by subsequent dilution with isotonic and isoionic solution to lower the EDTA concentration. Haemolysis is complete after 5 min; the cells are centrifuged, washed and resuspended in a solution of the same composition and osmolarity containing 4 mM MgCl₂, but no EDTA. The resealing process, carried out at 37°C, was complete in about 1 h. Measurements of the size distribution of the ghost cells in the hydrodynamically focusing Coulter Counter at varying field strengths in the orifice revealed that the ghost population is nearly uniform. The mean (modal) volume of the ghost cells was 110–120 $\text{μ}\text{m}{}^3$ when suspended in phosphate buffered NaCl solution. The apparent breakdown voltage was about 1.3 V.     

The characterization of energized and partially de-energized (respiration-independent) β-galactoside transport into escherichia coli

Unidirectional fluxes of [¹⁴C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN^?) conditions are analyzed kinetically.When the cells are energized, the value for $\text{V}$ influx is $\text{0.45 ± 0.01}\text{mM}$ internal concentration increment/s and $\text{K}{}_{\mathrm{t}}$ is $\text{0.26 ± 0.03}\text{mM}$. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is $\text{2 · 10}{}^3$.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of $\text{8.8 ± 0.2 · 10}{}^{\mathrm{?4}}\text{s}{}^{\mathrm{?1}}$.The transport in the presence of CN^? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O₂).The effects of CN^? are to lower the $\text{V}$ for influx and to change the efflux from a non-saturable to a saturable process with a value for $\text{K}{}_{\mathrm{t}}$ (60 mM) intermediate between that for energized efflux ($\text{> 250}\text{mM}$) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.     

Kinetic analysis of blood-brain barrier transport of amino acids

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of ¹⁴C-labelled amino acids and ³H₂O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15 s following injection. A brain uptake index (I_b) was determined from the ratio of ¹⁴C dpm in the brain tissue and the injection mixture divided by the same ratio for the ³H₂O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded K_m (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship $\text{P =}\text{V}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the I_b for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the ³H₂O reference 15 s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min^?1.     

A 3H-labelled trisaccharide from heparin as substrate for acetyl:CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase

The tetrasaccharide fraction obtained by gel chromatography after treatment of commercially available heparin with nitrous acid was reduced with NaB³H₄ and then hydrolysed with 2m trifluoracetic acid at 70° for 3 days. By gel chromatography and electrophoresis, the ³H-labelled trisaccharide 1 bearing an unsubstituted 2-amino-2-deoxy-d-glucosyl group in the non-reducing position was obtained (18% from the ³H-labelled tetrasaccharide). By sequential, enzymic degradation, the structure $\text{α-}\text{d}\text{-GlcN}\text{-(1→4)-β-}\text{d}\text{-GlcA}\text{-(1→4)-[1-}{}^3\text{H]aManol}$ was obtained for 1, which is a substrate for acetyl-CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase, an enzyme that is deficient in the Sanfilippo C syndrome. In human-skin fibroblasts, the pH optimum of acetyl transfer onto 1 was between pH 5.5 and 7.0, and dependent on the buffer. An apparent K_m for 1 of 0.14mM was found.     

Protease of adenovirus type 2. In vitro processing of core protein

Protease activity associated with temperature sensitive mutant ts3 of adenovirus type 2 was studied. This activity was induced only when ts3 was propagated at 33°C. By $\text{in vivo}$ and $\text{in vitro}$ experiments the enzyme was found to cleave main core polypeptide PVII to VII. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protease activity of ts3 was partially characterized. Phenylmethylsulfonyl fluoride (1mM) and SDS (0.5%) inhibited its activity completely. EDTA (10 mM) did not seem to inhibit its activity. Protease activity was completely abolished after 10 min. of incubation at 60°C. Autocatalytic cleavage of PVII to VII was not observed.     

Inhibition of urea synthesis by pent-4-enoate associated with decrease in N-acetyl-L-glutamate concentration in isolated rat hepatocytes

Pent-4-enoate at 0.1 to 1.0 mM strongly inhibited urea synthesis in isolated rat hepatocytes. Pent-4-enoate at the same concentrations markedly decreased concentrations of $\text{N-}\text{acetyl-}\text{L}\text{-glutamate}$, an essential activator of carbamoyl-phosphate synthase-I (EC 2.7.2.5), and the decrease was well parallel with the inhibition of urea synthesis by pent-4-enoate. This compound also lowered cellular concentrations of acetyl-CoA, a substrate of acetylglutamate synthase (EC 2.3.1.1). Pent-4-enoate in a dose of 1 mM did not significantly affect cellular concentrations of ATP, and had no direct effect on acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by pent-4-enoate is due to decrease in $\text{N-}\text{acetyl-}\text{L}\text{-glutamate}$ concentration and that the decrease is probably brought about by decreased rate of its synthesis due to the lowered concentration of cellular acetyl-CoA.     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

The zinc content of yeast alcohol dehydrogenase

Analyses for zinc in high specific activity preparations of yeast alcohol dehydrogenase (YADH) indicate a metal content of 1.8–1.9 moles of zinc per mole of enzyme subunit. This zinc content is observed for YADH prepared from Bakers yeast by recrystallization from Am₂SO₄ containing 1 mM EDTA, followed by chromatography on DE-52 and Sephadex-G-200. YADH obtained from Boehringer-Mannheim is characterized by a variable specific activity: preparations with Sp. Ac. = 380–400 U/mg contain 1.8–1.9 moles of zinc per mole of subunit. Dialysis of YADH against EDTA (pH 8.5, 25°, under N₂) reduces the specific activity and zinc content in an approximately linear fashion down to a Sp. Ac. = 150 U/mg, consistent with the preferential loss of a single, weakly bound zinc per subunit which is essential for catalytic activity. Dialysis of YADH against 1 mM ZnCl₂ (pH 6.5–8.5, 25°, under N₂) does not lead to an increase in the zinc content of the enzyme, indicating that under these conditions zinc does not bind adventitiously to YADH. Dialysis against 50 mM CoSO₄ (pH 5.5, 25°, under N2, 60–90 hr) leads to an exchange of ≈ 40% of the enzyme-bound zinc by cobalt. Our preparations of YADH are consistently characterized by a zinc content of ≈ 2 per subunit and we are unable to reduce the zinc content of YADH by dialysis against EDTA without a concomitant loss in enzyme activity, in contrast to reports of one zinc per subunit [Veillon, C. and Sytkowski, A.J., BBRC $\text{67}$: 1499 (1975); Vallee, B.L. and Hoch, F.L., Proc. Nat. Acad. Sci. USA $\text{41}$: 327 (1955)]. The findings reported here, together with the observed structural similarities between YADH and horse liver alcohol dehydrogenase [Jornvall, H., Woenckhaus, C. and Johnscher, G., Eur. J. Biochem. $\text{53}$: 71 (1975)], suggest a role for zinc at both a structural and catalytic site in YADH.     

Inactivation of phosphofructokinase by dialdehyde-ATP.

Rabbit muscle phosphofructokinase (PFK) is rapidly inactivated by a 2′,3′-dialdehyde derivative of adenosine triphosphate (dialdehyde-ATP). When allowed to react with 0.6 mm dialdehyde-ATP in 0.1 m borate buffer (pH 8.6) containing 0.2 mm EDTA and 0.5 mm dithiothreitol, PFK loses essentially all activity (99%) in 30 min. The modified PFK remains inactive following dialysis of the reaction mixture against sodium borate (pH 8.0) containing fructose diphosphate, EDTA, and dithiothreitol. Experiments with [¹⁴C]dialdehyde-ATP show that 99% inactivation of PFK corresponds to incorporation of 3 to 4 mol of the ATP analog per PFK protomer. The inactivation of PFK with dialdehyde reagent is not caused by dissociation of the 340,000 M_r, tetramer to the 170,000 M_r dimer, as determined by analytical ultracentrifugation. Adenosine diphosphate or ATP protect PFK from inactivation by dialdehyde-ATP at pH 8.6, but fructose 6-phosphate, cyclic 3′,5t$\text{-}\text{?}$adenosine monophosphate, or fructose diphosphate, which protect PFK from modification by pyridoxal phosphate, provide little protection from inactivation. Amino acid analyses of dialdehyde-inactivated PFK and of a control sample of the enzyme were compared following reaction of each with 2,4-dinitrofluorobenzene. The results show that three or four lysine residues per PFK protomer are modified by dialdehyde-ATP. Additional data indicate that these lysine residues react with dialdehyde-ATP to form dihydroxymorpholine-like adducts rather than Schiff bases.     

Analysis of chlorophyll fluorescence induction curves in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea as a function of magnesium concentration and NADPH-activated light-harvesting chlorophyll ab-protein phosphorylation

The effect of Mg²⁺ concentration and phosphorylation of light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ on various chlorophyll fluorescence induction parameters of isolated pea thylakoids has been studied. (1) Lowering the Mg²⁺ concentration from 3 to 0.4 mM decreases only the variable fluorescence (F_v) and the area above the induction curve while at the same time increasing the slow exponential component of the rise (β_max). (2) A further decrease in Mg²⁺ concentration from 0.4 to 0 mM decreases the initial (F₀) fluorescence level such that the ratio $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ increases slightly as does the area above the induction curve and β_max. (3) Thylakoid membranes, phosphorylated at 5 mM Mg²⁺, show an equal decrease in F_v and F₀, no change in the area above the induction curve and an increase in β_max. At 2 mM Mg²⁺, however, phosphorylation induced a more extensive quenching of F_v so that the $\text{F}{}_{\mathrm{<mtext>v</mtext>}}\text{F}{}_{\mathrm{<mtext>m</mtext>}}$ ratio was lowered and the area above the induction curve decreased while β_max increased. (4) When phosphorylated membranes were subsequently suspended in an Mg²⁺-free medium the effect on F₀ due to phosphorylation was found to be additive to that due to the absence of Mg²⁺. The effect of membrane phosphorylation on fluorescence is discussed in relation to the control of excitation energy distribution and shows that different mechanisms operate depending on the background Mg²⁺ levels. At high Mg²⁺ the phosphorylation seems to affect the absorption cross-section of Photosystem II while at lower Mg²⁺ levels there is an additional effect of increased spillover from Photosystem II to I.     

Requirement of different ionic concentrations for optimal translation of α and β globin mRNA in Krebs II ascites cell-free system

The translation of rabbit α globin mRNA in a Krebs II ascites cellfree system was more dependent upon the K⁺ concentration than rabbit β globin mRNA. The optimal KCl concentration was approximately 70 mM for the synthesis of the α chain and between 80 and 90 mM for that of the β chain. With CH₃ CO₂K the optimum concentration for α chain synthesis was also 70 mM but the optimum for the β chain synthesis was not sharp any more and ranged from 70 mM to over 110 mM. In the range of the optimal Mg²⁺ concentration for the α and β globin chain synthesis the $\text{α}\text{β}$ ratio decreased when the Mg²⁺ concentration increased. In the presence of DTT and EDTA the optimal KCl concentration for both α and β globin chain synthesis decreased.     

Phosphate transport in Neurospora: Kinetic characterization of a constitutive,low-affinity transport system

Log-phase cells of Neurospora crassa, grown in standard minimal medium, possess an energy-dependent transport system for inorganic phosphate, with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ (at pH 5.8) of 0.123 mM and a J_max of 1.64 mmoles/l cell water per min. Like the PO₄^3? transport system in yeast, the Neurospora system is stimulated by high intracellular K⁺. In addition, it is inhibited by high extracellular salt concentrations, an effect which may be related to the known depolarization of the Neurospora plasma membrane at high salt concentrations.The most striking property of the system is its strong dependence upon the extracellular pH. From pH 4.0 to pH 7.3, the J_max remains essentially constant but the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increases nearly 400-fold, from 0.01 to 3.62 mM. The increase cannot be accounted for by a single system with a preference for H₂PO₄^? (which would show only a 3-fold increase in apparent $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ over this pH range) nor by two systems with different affinities and pH optima (which would display nonlinear double-reciprocal plots at intermediate pH values). It can be explained, however, by a model in which OH^? or H⁺ is assumed to act as a modifier of the transport system, altering its affinity for substrate.