Identification of the altered subunit in the inactive F1ATPase of an Escherichia coli uncA mutant.

ATPase activity was restored to the inactive coupling factor, F₁ATPase, of $\text{Escherichia coli}$ strain AN120 ($\text{uncA401}$) by reconstitution of the dissociated complex with an excess of wild-type α subunit. Large excesses of α gave the highest levels of activity. The other subunits which are required for the reconstitution of ATPase activity, β and γ, did not complement the mutant enzyme. These results indicate that the α polypeptide of the AN120 ATPase is defective.     

A novel subunit separation procedure for cryptomonad phycoerythrin 545.

A method to separate phycoerythrin 545, isolated from the cryptomonad alga, $\text{Rhodomonas}\text{lens}$, into two subunits has been developed. The method uses no denaturants (urea, guanidine, detergent) but relies on dissociation of the dimeric protein and subsequent aggregation of the β subunit at pH 3.0. The absorption spectra and amino acid composition of the subunits are presented. The spectra of the α subunit was red-shifted relative to β in both pH 3.0 buffer and in acidic 8.0 M urea.     

D2O-alanine exchange reactions catalyzed by alanine racemase and glutamic pyruvic transminase

NMR studies in D₂O (>90%) reveal that Alanine Racemase (5.1.1.1.) from $\text{B. subtilis}$ catalyzes the exchange of the α hydrogen of $\text{D}$- and $\text{L}$-alanine with D₂O. Glutamic Pyruvic Transaminase (2.6.1.2.) and Glutamic Oxaloacetic Transaminase (2.6.1.1.) catalyze the exchange of α and β hydrogens of $\text{L}$-alanine. The rates of exchange of α and β hydrogens appear to be of the same order of magnitude. The transaminase catalyzed exchange is enhanced by catalytic amounts of pyruvate. The side chain of $\text{L}$-alanine is held more rigidly at the active site of transaminase so that the planar conjugated system can be extended to include the α and β carbons. A generalized mechanism is proposed for the action of pyridoxal phosphate dependent transaminases which extends Braunstein and Snell mechanism to include the structures which contribute to the labilization of β hydrogens of amino acids by the transaminases that have been studied.     

The use of affinity elution from Blue Dextran Sepharose by yeast tRNA2Val in the complete purification of the cytoplasmic valyl-tRNA synthetase from Euglena gracilis

Two distinct monomers, α and β participate in the structures of diffe$\text{r}$ ent oligomers of $\text{Neurospora crassa}$ glutamine synthetase (EC 6. 3. 1. 2). In ammonium-limited cultures a tetrameric form composed mainly of α monomers was found. In excess of nitrogen an octameric form composed mainly from β monomers is the predominant oligomeric state. The presence of both monomers was observed in intermediate oligomeric forms.     

Adenosine kinase from Trypanosoma cruzi

Adenosine kinase was demonstrated in the soluble fraction of $\text{Trypanosoma}\text{cruzi}$. Magnesium is required for activity. ATP and GTP are efficient phosphate donors while $\text{p}\text{-}\text{nitrophenylphosphate}$ is without activity. The pH optimum is high (8.0), it is heat labile and is stabile to freezing (?20° or ?80°). It is substrate inhibited, does not survive dialysis and its stability to gel filtration is enhanced by the presence of ATP or GTP. Time curves are parabolic only if the enzyme is preincubated with ATP (or GTP); sigmoid when preincubated with adenosine.     

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.     

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

The three major subunits (α, β and γ) of the coupling factor, F₁ ATPase, of $\text{Escherichia coli}$ were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgCl₂. A mixture containing α, β and γ regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with α plus β. The reconstituted ATPase had a K_m of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000.     

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

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

An active-site-directed irreversible inhibitor of delta 5-3-ketosteroid isomerase

The α and β isomers of spiro-3-oxiranyl-5α-androstan-17β-ol were tested as possible inhibitors of Δ⁵-3-ketosteroid isomerase of $\text{Pseudomonas}\text{testosteroni}$. The β-oxirane causes a first-order irreversible inactivation of the enzyme and shows saturation kinetics (K_I, 17 μM). Protection against inactivation is exhibited by 19-nortestosterone, a competitive inhibitor of the isomerase. Although the α-oxirane was found to be a good reversible inhibitor (K_i, 21 μM), prolonged incubation with it failed to produce any inactivation of the isomerase. The results obtained are consistent with the presence of a nucleophilic group situated near the 3-keto group of the substrate in the enzyme-steroid complex.     

Immunoreactivity of subunits of the (Na+ + K+)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of Na⁺, Mg²⁺, and [$\text{γ-}{}^{32}\text{P}\text{]}\text{ATP}$ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

Preliminary x-ray diffraction studies of EcoRI restriction endonuclease-DNA complex

A complex between EcoRI restriction endonuclease and cognate DNA fragment, 5′-G-A-A-T-T-C C-T-T-A-A-G-5′, has been crystallized. The space group is P42₁2 with $\text{a = b = 183.2}\text{A}\text{?}\text{, c = 49.7}\text{A}\text{?}\text{, α = β = γ = 90 °}$. The unit cell contains four enzyme monomers plus two duplex DNA fragments in an asymmetric unit. High quality crystals of the enzyme alone have also been obtained.     

Thiol modulation of the chloroplast protonmotive ATPase and its effect on photophosphorylation

Thiol modulation of the chloroplast protonmotive ATPase (CF₀-CF₁) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall $\text{P}\text{2}\text{e}$ ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in $\text{P}\text{2}\text{e}$ ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH₄Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in $\text{P}\text{2}\text{e}$ ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and $\text{P}\text{2}\text{e}$ ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF₀-CF₁ increased the observed Michaelis constant for ADP (K_m(ADP)) and the apparent maximum rate (V_app of photophosphorylation by the same factor, so that ratio $\text{V}{}_{\mathrm{<mtext>app</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was not altered. $\text{V}{}_{\mathrm{<mtext>app</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF₀-CF₁ is presented to explain the dependence of α on ΔpH. Thiol modulation of CF₀-CF₁ is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process.     

The hydrophobic nature of the pig intrinsic factor receptor in the intestine

The pig intestinal intrinsic factor receptor has been isolated and dissociated into its α and β subunits. The β subunit was found to be more hydrophobic than the α subunit. In a detergent solution only the α subunit was accessible to digestion with papain. The whole isolated receptor was introduced into artificial single bilayer liposomes where is apparently was randomly oriented. Liposomes containing the receptor were digested with papain and the polypeptide segments that stayed in the lipid fraction were extracted and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Four species were found with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ values of 23 000, 45 000, 70 000 and 86 000.     

NADP-malic enzyme from maize leaf: regulatory properties.

The regulatory properties of purified maize leaf NADP-malic enzyme (EC 1.1.1.40) were studied at three different pHs and the following results were obtained. (a) At pH 7.5 enzyme activity reaches a maximum at 0.4–0.8 mm malate depending on the Mg²⁺ concentration, and higher levels of malate result in marked substrate inhibition; with increasing pH the degree of substrate inhibition is reduced to where at pH 8.4 little or no inhibition is observed. (b) The inhibitory effect of malate is more pronounced at 1 mm Mg²⁺ than at 5–10 mm Mg²⁺ in the pH range of 7.5 to 8.4; a plot of enzyme activity vs Mg²⁺ concentration at 3 mm malate follows Michaelis-Menten kinetics at both pH 7.5 and 8.4; the apparent affinity of the enzyme for Mg²⁺ at pH 8.4 was threefold greater than that at pH 7.5. (c) The activity of NADP-malic enzyme decreases as the ratio of $\text{NADPH}\text{NADP}$ increases, and this effect is enhanced at lower pH. (d) Various α-keto acids including glyoxylate, oxaloacetate, and α-ketoglutarate inhibit NADP-malic enzyme activity, whereas HCO₃^?, pyruvate, and other organic acids, sugar phosphates, and amino acids have little or no effect on the activity of the enzyme. Based on these experimental findings, the regulatory properties of maize leaf NADP-malic enzyme are discussed with respect to its key role in net CO₂ fixation in maize bundle sheath chloroplasts during C₄ photosynthesis.     

Kinetic considerations for the regulation of adenosine and deoxyadenosine metabolism in mouse and human tissues based on a thymocyte model

Metabolic regulation at a branch point may be determined primarily by relative enzyme activities and affinity for common substrate. Adenosine and deoxyadenosine are both phosphorylated and deaminated and their metabolism was studied in intact mouse thymocytes. From kinetic considerations of two activities competing for a common substrate, the deamination:phosphorylation ratio, $\text{v}{}^{\mathrm{<mtext>d</mtext>}}\text{v}{}^{\mathrm{<mtext>k</mtext>}}$, at high nucleoside concentration, $\text{[}\text{S}\text{]?∞}$, is equal to $\text{V}{}^{\mathrm{<mtext>d</mtext>}}\text{V}{}^{\mathrm{<mtext>k</mtext>}}$, or 34 and 1090 for adenosine and deoxyadenosine, respectively. At low substrate concentrations, $\text{[}\text{S}\text{]?0}$, $\text{v}{}^{\mathrm{<mtext>d</mtext>}}\text{v}{}^{\mathrm{<mtext>k</mtext>}}$ is equal to $\text{V}{}^{\mathrm{<mtext>d</mtext>}}\text{K}{}^{\mathrm{<mtext>k</mtext>}}{}_{\mathrm{<mtext>m</mtext>}}\text{V}{}^{\mathrm{<mtext>k</mtext>}}\text{K}{}^{\mathrm{<mtext>d</mtext>}}{}_{\mathrm{<mtext>m</mtext>}}$, or 0.7 and 285 for adenosine and deoxyadenosine, respectively. The analysis was extended to other mouse and human tissues by measurement of adenosine kinase, deoxyadenosine kinase and adenosine deaminase activities. All tissues were found to preferentially deaminate deoxyadenosine. Three tissue types were apparent with respect to adenosine metabolism: those which preferentially phosphorylate adenosine at all concentrations, those which switch from phosphorylation to deamination between low and high adenosine concentration and those for which deamination is quantitatively important at all concentrations. Lymphoid tissues are representative of the latter category. The kinetic approach we describe offers a means of predicting nucleoside metabolism over a range of concentration which may be technically difficult to otherwise measure. The phosphorylation of adenosine and deoxyadenosine was also studied in intact thymocytes in the presence of adenosine deaminase inhibitors. The rate of deoxyadenosine phosphorylation was unaffected by coformycin or EHNA, whereas adenosine phosphorylation decreased with increasing substrate concentrations to 18% the rate in the absence of adenosine deaminase inhibitors.     

Glutathione S-transferases from the New Zealand grass grub,Costelytra zealandica Their isolation and characterization and the effect on their activity of endogenous factors

Isolation of glutathione $\text{S-}\text{transferase}$ from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates.Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 23,700 and the pI 5.9 enzyme one of subunit $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 22,500.A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme.     

Effect of sodium deoxycholate on 5′-nucleotidase

The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes

Using a citrate-EDTA buffer as an anticoagulant it was possible to isolate intact haemocytes from the insect, Blaberus craniifer, without causing extensive degranulation and subsequent clotting. A haemocyte lysate from this insect contained prophenoloxidase (proPO), which could be activated by β 1,3-glucans. The activation process was dependent upon Ca²⁺ ions and seemed to occur by a limited proteolysis, since several serine protease inhibitors such as soybean trypsin inhibitor, benzamidine and $\text{p-}\text{nitrophenyl}\text{-p′-}\text{guanidobenzoate}$ blocked convertion of proPO to the active enzyme. Treatment of proPO with urea or heat also caused proPO activation but probably without the intervention of serine proteases, since the protease inhibitors used failed to block the activation. Within the haemocyte lysate, several endopeptidases were present, which were enhanced in activity by prior treatment with β 1,3-glucans. These endopeptidases were inhibited in activity when the haemocyte lysate was incubated with benzamidine prior to the addition of β 1,3-glucan. This provides further indications that the activation of proPO involves a limited proteolytic attack. The active phenoloxidase enzyme became strongly bound to foreign surfaces and this phenomenon may assist in providing opsonic properties for the proPO cascade.     

Modulation of lyso-platelet-activating factor:acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions

The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca²⁺ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca²⁺. The effect of Ca²⁺ was on the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of the enzyme for the substrate acetyl-CoA without showing any significant effect on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca²⁺ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca²⁺, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca²⁺ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation.