Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein

Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as "des-1-25-fructose-1,6-bisphosphatase." A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6-bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25-fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP-binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule.     

The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin

The functional interactions of the retinal G protein, transducin, with the cyclic GMP phosphodiesterase (PDE) have been examined using the different purified subunit components of transducin and the native and trypsin-treated forms of the effector enzyme. The limited trypsin treatment of the PDE removes the low molecular weight gamma subunit (Mr approximately 14,000) of the enzyme, yielding a catalytic moiety comprised of the two larger molecular subunits (alpha, Mr approximately 85,000-90,000; beta, Mr approximately 85,000-90,000), which is insensitive to the addition of either the pure alpha T.GTP gamma S species or the pure beta gamma T subunit complex. However, the addition of the pure alpha T.GDP species to the trypsin-treated PDE (tPDE) results in a significant (90-100%) inhibition of the enzyme activity. This inhibition can be reversed by excess beta gamma T, suggesting that the holotransducin molecule does not (functionally) interact with the tPDE. However, the inhibition by alpha T.GDP is not reversed by the alpha T.GTP gamma S complex, over a range of [alpha T.GTP gamma S] which elicits a marked stimulation of the native enzyme activity, suggesting that the activated alpha T species does not effectively bind to the tPDE. The alpha T.GDP complex also is capable of inhibiting the alpha T.GTP gamma S-stimulated cyclic GMP hydrolysis by the native PDE. This inhibition can be reversed by excess alpha T.GTP gamma S, as well as by beta gamma T, indicating that the binding site for the activated alpha T species is in close proximity and/or overlaps the binding site for the alpha T.GDP complex on the enzyme. Overall, these results are consistent with a scheme where (a) both the small and larger molecular weight subunits of PDE participate in alpha T-PDE interactions, (b) the activation of PDE by the alpha T.GTP gamma S (or alpha T.GTP) species does not result in the complete dissociation of the gamma subunit from the enzyme, and (c) the deactivation of this signal transduction system results from a direct interaction between the alpha T.GDP species and the catalytic moiety of the effector enzyme.     

A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits.

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

The mechanism of conversion of rat liver xanthine dehydrogenase from an NAD+-dependent form (type D) to an O2-dependent form (type O).

Rat liver xanthine dehydrogenase, type D, has been isolated directly from crude extracts as an antibody complex and its properties compared with those of two oxidase forms of the enzyme, heat-treated type O and trypsin-treated type O, also isolated as antibody complexes. The type D antibody complex displays electron acceptor specificities and electron paramagnetic resonance properties characteristic of an NAD⁺-dependent dehydrogenase whereas the trypsin-treated type O complex behaves as an O₂-utilizing oxidase. The heat-treated type O complex displays intermediate behavior. After electrophoresis in dodecyl sulfate-urea-acrylamide gels, type D and heated type O enzymes show single polypeptide bands, each of approximately 150,000 molecular weight. The trypsinized type O also shows one major band but with an approximate molecular weight of 130,000. Purified type D enzyme, when proteolytically treated, is converted to an oxidase with increased mobility on polyacrylamide gels. The 150,000 molecular weight subunit is cleaved into smaller subunits during proteolysis. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) converts the type D enzyme, whether isolated as the purified enzyme or as the immune precipitate, to type O enzyme in a time-dependent manner. Titration of type D and the two type O antibody complexes with 5,5′-dithiobis-(2-nitrobenzoic acid) reveals that type D and heated type O each has approximately 28 reactive sulfhydryls, whereas the trypsinized type O has only 8 such groups. Many of the free sulfhydryls are vicinal and form disulfide bonds during the conversion to an oxidase by this reagent. Unproteolyzed preparations of type O rat liver enzyme and milk xanthine oxidase are converted to type D enzymes by treatment with dithiothreitol. The converted enzymes display electron acceptor specificities and epr properties characteristic of an NAD⁺-dependent dehydrogenase molecule.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Characterization of purified guanine aminohydrolase.

Guanine aminohydrolase (GAH) (E.C. 3.5.4.3) was purified by affinity chromatography on 9-(p-β-aminoethoxyphenyl)guanine-Sepharose to a specific activity of 35.5 units/mg. The molecular weight of the enzyme was estimated to be 110,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) showed that the enzyme was composed of subunits with molecular weights of approximately 52,000. Data from SDS-gel electrophoresis in a discontinuous buffer system and from isoelectric focusing in the presence of 8-m urea indicated that more than one type of subunit were present. This was consistent with multiple forms of the native enzyme seen by electrophoresis and isoelectric focusing in polyacrylamide gels. The isoelectric points for the different forms of GAH were in the range of 4.65–4.85. Amino acid analyses showed cysteine to be the minimum amino acid and gave a calculated molecular weight for GAH of 53,016 when the assumption that there were four cysteines per subunit was made. Guanine, 8-azaguanine, and 6-thioguanine served as substrates for the enzyme but 3-deazaguanine, a potent competitive inhibitor of GAH, did not. Fluoride ion inhibited the enzyme in a noncompetitive manner, and this inhibition decreased as pH increased. Variation of the kinetic parameters with pH suggested that hydroxide ion might be the second substrate and that a functional group on the enzyme with a pK_a near 5.6 was involved in the reaction. The enzyme was inactivated by treatment with p-hydroxymercurobenzoate and by photooxidation in the presence of rose bengal. Two plausible mechanisms are proposed for the reaction catalyzed by GAH.     

Trypsin cleavage of the α-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable) nucleotide binding in beef heart mitochondrial F₁-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103–7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the α-subunit of F₁ is altered by the mild trypsin treatment, whereas no change was detected in β-, γ-, δ- or ?-subunits. Binding of ADP to the trypsin-treated F₁ was compared to binding to control enzyme over a range of 0–40 μM ADP in a 30 min incubation period. There was no difference between the two enzymes, K^ADP_d in Mg²⁺-containing buffer was about 2 μM in each. Since the tight (nonexchangeable) sites are abolished in trypsin-treated F₁, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the α-subunit on β-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the α-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F₁ but leaves partial functions intact.     

Purification and some properties of guanidinoacetate amidinohydrolase produced by Corynebacterium sp.

Guanidinoacetate amidinohydrolase (EC 3.5.3.2) was purified from Cornebacterium sp. grown in a medium supplemented with guanidinoacetate, and some of its properties were investigated.The molecular weight of the enzyme was estimated to be 150,000 by gel filtration. SDS-polyacrylamide gel electrophoresis showed a single subunit component with a molecular weight of 38,000, suggesting that the enzyme is composed of four identical subunits. The isoelectric point of the enzyme was pH 5.8.The enzyme showed optimum activity at pH 9.0–9.5 and was stable at pH 6.0–10.5. 3-Guanidinopropionate and 4-guanidinobutyrate were respectively hydrolyzed 32% and 5% as fast as guanidinoacetate. The apparent K_m for guanidinoacetate was 16 mM. Incubation of the enzyme by o-phenanthroline or 8-hydroxyquinoline resulted in almost complete inactivation. The activity of the inactivated enzyme was restored by incubation with Zn²⁺. p-Chloromercuribenzoic acid and iodine effectively inhibited the enzyme activity. Glycine was a competitive inhibitor, and n-alkyl amines such as n-octylamine, n-decylamine and n-dodecylamine were uncompetitive inhibitors.     

KINETIC ASSESSMENT AND EFFECT ON DEVELOPMENTAL PHYSIOLOGY OF A TRYPSIN INHIBITOR FROM Eugenia jambolana (JAMBUL) SEEDS ON Helicoverpa armigera (HÜBNER)

A trypsin inhibitor was purified from the seeds of Eugenia jambolana (Jambul) with a fold purification of 14.28 and a yield recovery of 2.8%. Electrophoretic analysis of E. jambolana trypsin inhibitor (EjTI) revealed a molecular weight of approximately 17.4 kDa on 12% denaturing polyacrylamide gel electrophoresis with or without reduction. EjTI exhibited high stability over a wide range of temperatures (4–80 °C for 30 min) and pH (3.0–10.0) and inhibited trypsin‐like activities of the midgut proteinases of fourth instar Helicoverpa armigera larvae by approximately 86%. Feeding assays containing 0.05, 0.15, and 0.45 (% w/w) EjTI on functionally important fourth‐instar larvae indicated a dose‐dependent downfall in the larval body weight as well as on extent of survival. The nutritional analysis suggests that EjTI exerts toxic effects on H. armigera. Dixon plot analysis revealed competitive inhibition of larval midgut proteinases by EjTI, with an inhibition constant (K_i) of approximately 3.1 × 10^?9 M. However, inhibitor kinetics using double reciprocal plots for trypsin inhibition demonstrated a mixed inhibition pattern. These observations suggest the potential of E. jambolana trypsin inhibitor protein in insect pest management.     

Folic acid effects on glycoprotein-galactosyltransferase: a re-assessment

Prostaglandin Δ¹³-reductase was purified extensively by ammonium sulfate fractionation, and DEAE-Sephadex-, hydroxylapatite-, and phosphocellulose chromatography. Enzyme activity was followed by radioimmunoassay with the use of an antiserum directed toward 15-keto-prostaglandin F_2α and antibodies directed toward 13, 14 dihydro-15-keto-prostaglandin F_2α. The purified enzyme used NADPH as a cofactor much more effectively than NADH. It specifically reduced 15-keto-prostaglandins but not 15-hydroxy-prostaglandins. The enzyme was inhibited by p-chloromercuribenzoate. It had a relatively broad pH optimum (pH 7.4 to pH 8.5), and has a molecular weight estimated to be 70,000 to 80,000.     

Purification and properties of 5′-nucleotidase from alkalophilic Bacillus No. C-3

5′-Nucleotidase (EC 3. 1. 3. 5) from alkalophilic Bacillus no. C-3 was purified to homogeneity. The molecular weight of the enzyme was 80,000 by gel filtration. The optimum pH for the activity was 9.5, and the enzyme was stable at pH 9.5–10.5 in a buffer containing 10 mM 2-mercaptoethanol. Substrate specificity study revealed that the enzyme acted on 5′-AMP strongly, on several 5′-nucleotides and ADP to a certain extent, but not on 3′-nucleotides, 2′-nucleotides, p-nitrophenyl phosphate, or ATP. The K_m value for 5′-AMP was 3.0 × 10⁻⁴ M. The enzyme required no divalent cation for its activity. The enzyme was inhibited by borate and arsenite ions but not by 1 mM EDTA.     

THE INFLUENCE OF A 21 kDa KUNITZ‐TYPE TRYPSIN INHIBITOR FROM NONHOST MADRAS THORN,Pithecellobium dulce,SEEDS ON H. armigera (HÜBNER) (LEPIDOPTERA: NOCTUIDAE)

A trypsin inhibitor purified from the seeds of the Manila tamarind, Pithecellobium dulce (PDTI), was studied for its effects on growth parameters and developmental stages of  Helicoverpa armigera. PDTI exhibited inhibitory activity against bovine trypsin (～86%; ～1.33 ug/ml IC₅₀). The inhibitory activity of PDTI was unaltered over a wide range of temperature, pH, and in the presence of dithiothreitol. Larval midgut proteases were unable to digest PDTI for up to 12 h of incubation. Dixon and Lineweaver–Burk double reciprocal plots analysis revealed a competitive inhibition mechanism and a K_i of ～3.9 × 10^?8 M. Lethal dose (0.50% w/w) and dosage for weight reduction by 50% (0.25% w/w) were determined. PDTI showed a dose‐dependent effect on mean larval weight and a series of nutritional disturbances. In artificial diet at 0.25% w/w PDTI, the efficiency of conversion of ingested food, of digested food, relative growth rate, and growth index declined, whereas approximate digestibility, relative consumption rate, metabolic cost, consumption index, and total developmental period were increased in larvae. This is the first report of antifeedant and antimetabolic activities of PDTI on midgut proteases of  H. armigera.     

Limited proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solublized coupling factor with chymotrypsin rapidly produced an A′-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the ATPase enzyme. Secondary chymotryptic cleavage yielded an A″-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at ?20°C in the presence of 4 mM MgCl₂ with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4°C in the presence or absence of MgCl₂ both decreased ATPase activity and generated A′-type alpha subunit species. Since presence of phenylmethylsulfonyl fluoride prevented these changes, an unknown protease was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparently located within the same small segment of alpha subunit polypeptide chain.     

Structural, Functional, and Evolutionary Analysis of Ribulose-1,5-Bisphosphate Carboxylase from the Chromophytic Alga Olisthodiscus luteus

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg²⁺ have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO₃ and Mg²⁺ for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO₂ and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase-catalyzed CO₂ fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site.     

Studies on aspartase. III. Alteration of enzymatic properties upon trypsin-mediated activation.

Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.     

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

Partial resolution of the enzymes catalyzing photophosphorylation. XV. Approaches to the active site of coupling factor I.

1. Prolonged treatment of coupling factor I (CF1) from spinach chloroplasts with trypsin free of chymotrypsin yielded an active ATPase. The isolated preparation showed only two polypeptide chains (mol wt 55,000 to 60,000) on acrylamide gels run in the presence of sodium dodecyl sulfate. The three smaller subunits of CF1 were not detectable. The preparation no longer served as a coupling factor for photophosphorylation in either EDTA- or silicotungstate-treated chloroplasts. 2. An antiserum prepared against coupling factor I from chloroplasts inhibited the ATPase activity of the trypsin-treated CF1. In contrast, antisera prepared against the two individual (denatured) subunits did not inhibit the ATPase activity when tested either alone or together, although each interacted with the trypsin-treated protein, forming precipitin lines in Ouchterlony plates. 3. The trypsin-treated enzyme was still cold-labile, showing that the three smaller subunits are not required for this property. However, the enzyme was no longer sensitive to the natural inhibitor protein which is one of its subunits (subunit epislon), but was still sensitive to inhibition by the flavonoid quercetin. 4. Two equivalents of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole were sufficient to inhibit about 80% of the ATPase activity of the coupling factor, irrespective of whether it contained two of five subunits. The inhibition was completely reversed by dithiothreitol. 5. Triated 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole was prepared. Treatment of the coupling factor with this tritium-labeled inhibitor followed by electrophoresis on acrylamide gels revealed that most of the radioactivity was incorporated into the beta subunit of the enzyme (molecular weight 56,000).     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

Proton flux through the chloroplast ATP synthase is altered by cleavage of its gamma subunit

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.