An alternating site sequence for oxidative phosphorylation suggested by measurement of substrate binding patterns and exchange reaction inhibitions.

Catalysis by beef heart submitochondrial particles of the medium Pi in equilibrium HOH, Pi in equilibrium ATP, and the ATP in equilibrium HOH exchanges is strongly inhibited while the ATPase and intermediate Pi in equilibrium HOH exchange are accelerated when medium ADP is removed by pyruvate kinase action. Arsenate readily blocks completely the Pi in equilibrium ATP and medium Pi in equilibrium HOH exchange reactions, but not the ATP in equilibrium HOH exchange reaction. The residual ATP in equilibrium HOH exchange in presence of arsenate is inhibited by 2,4-dinitrophenol.

Evidence for energy-dependent change in phosphate binding for mitochondrial oxidative phosphorylation based on measurements of medium and intermediate phosphate-water exchanges.

Characteristics of the exchange reactions catalyzed by beef heart submitochondrial particles give new insight into energy transducing steps of oxidative phosphorylation. The uncoupler-insensitive portion of the total Pi in equilibrium HOH exchange in presence of ATP, ADP, and Pi is the intermediate Pi in equilibrium HOH exchange, that is the exchange occurring with Pi formed by hydrolysis of ATP prior to release of Pi from the catalytic site. The exchange of medium Pi with HOH is as sensitive to uncouplers as the Pi in equilibrium ATP exchange and net oxidative phosphorylation, demonstrating a requirement of an uncoupler-sensitive energized state, probably a transmembrane potential or proton gradient, for bringing medium Pi to the reactive state.

Dynamic reversal of enzyme carboxyl group phosphorylation as the basis of the oxygen exchange catalyzed by sarcoplasmic reticulum adenosine triphosphatase.

Millisecond mixing and quenching experiments demonstrate an apparent t1/2 for the labeling of phosphorylated sarcoplasmic reticulum ATPase by 32Pi at pH 6 and 30 degrees C of 30 to 40 ms. Under the same conditions, the rate of exchange of water oxygens with inorganic phosphate (Pi) is about 40 mol of H2O exchanged with Pi per 10(6) g of protein per s. Theoretical equations are developed for the expected 32P-labeling pattern given various comparative rates of flux between Pi and the Michaelis complex and between the Michaelis complex and phosphorylated enzyme.

Regional intralymphatic infusion (ILI) of irradiated tumor cells with evidence of distant effects.

A case of canine lymphoma with radiographically-documented involvement of the para-aortic nodes is reported. Intralymphatic infusions (ILI) of cultured irradiated autochthonous tumor cells to remote lymph node bearing areas were associated with a dramatic initial shrinkage of the para-aortic lymphadenopathies. Three ILI timing schedules were used consecutively during a course of 10 treatments, allowing a comparison of responses in the same animal.

Demonstration of a transitory tight binding of ATP and of committed P(i) and ADP during ATP synthesis by chloroplasts.

Rapid mixing, quenching, and filtration experiments with chloroplast thylakoid membranes, with energization by acid-base transition, demonstrate that an ATP tightly bound to the isolated membranes is a transient intermediate in the catalytic sequence for ATP synthesis. The experiments also show that most of the P(i) and ADP bound at a catalytic site is committed to ATP formation without interchange with medium P(i) or ADP. Other results give evidence that upon energization, the tightly bound ADP that is detectable in isolated thylakoid membranes or coupling factor ATPase is rapidly released to the medium from a catalytic site.

Facile alkylation of methionine by benzyl bromide and demonstration of fumarase inactivation accompanied by alkylation of a methionine residue.

Benzyl bromide is a selective alkylator of sulfur nucleophiles including methionine and cysteine. Only the mercaptide ion is a more efficient nucleophile than is the sulfur ether of methionine. Alkylation rates relative to methionine are 200: less than or equal to 0.

Rapid transfer of oxygens from inorganic phosphate to glutamine catalyzed by Escherichia coli glutamine synthetase.

Measurements are reported on certain isotopic fluxes during the net conversion of glutamine, ADP and Pi to glutamate, NH3, and ATP by Escherichia coli glutamine synthetase (adenylylated form, Mn2+ activated) in presence of a hexokinase/glucose trap to remove the ATP formed during the reaction. The results show that the transfer of oxygens from Pi to glutamine is the most rapid of the measured isotopic interchanges, over five oxygens from Pi being transferred to glutamine for each glutamate formed by net reaction. Under similar conditions, the oxygen transfer from Pi to glutamate, was stimulated somewhat by an increase in the glutamate concentration but inhibited by an increase in the ammonia concentration.

Probes of initial phosphorylation events in ATP synthesis by chloroplasts.

Rapid mixing and quenching techniques have been used with chloroplasts activated by an acid-base transition or by light to assess the nature and characteristics of the substances initially labeled by inorganic [32P]phosphate during ATP synthesis. With light-activated chloroplast fragments, but not with acid-base-activated preparations, an initial rapid labeling of a small amount of ADP is observed. With the acid-base activated preparations a slower continued labeling of ADP occurs that is uncoupler-sensitive, that does not proceed via [gamma-32]ATP of the medium and for which medium ADP furnishes the AMP moiety.

Secondary in vitro lymphocyte-proliferative responses to syngeneic plasma cell tumors.

Two characteristics of immune responses to weakly immunogenic plasma cell tumors were demonstrated in this study. (a) Elevated lymphoproliferative responses following in vivo inoculation of sublethal doses of plasma cell tumors were detected by a mixed lymphocyte-tumor interaction (MLTI) assay. Specificity of elevated MLTI responses correlated with resistance to in vivo tumor challenge.

Stimulation of lymphoid cells from normal and immune mice by syngeneic BALB/c plasma cell tumors.

Lymphoid cells from normal and immunized BALB/c mice could be stimulated in vitro by syngeneic PCT contrasted with an absence of response to a number of other tumors. Maximal responses of normal cells to PCT were found to occur 5 days after the initiation of the cultures at an optimal responding:stimulation cell ratio of 1:2. MLTI activity of normal cells could not be blocked or enhanced by PCT myeloma protein products indicating that MLTI reactivity was directed against non-idiotypec cell surface determinants.

A model for conformational coupling of membrane potential and proton translocation to ATP synthesis and to active transport.

Acceptance of a membrane potential and/or a proton gradient as a possible means of transmitting energy from oxidations to ATP synthesis rests in part on a satisfactory hypothesis for how the potential or proton gradient could drive ATP synthesis. Recognition that energy input may drive ATP synthesis by change in binding of reactants at the catalytic site has led to the suggestions presented in this paper. These are that in oxidative phosphorylation and photophosphorylation, the requisite conformational changes may be coupled to exposure of charged groups to different sides of the membrane.

Oxygen exchange in the gamma-phosphoryl group of protein-bound ATP during Mg2+-dependent adenosine triphosphatase activity of myosin.

When ATP binds to myosin in the presence of Mg2+ there follows a rapid cleavage reaction to yield a myosin-product complex whose breakdown is rate-limiting in the overall adenosine triphosphatase reaction at 21 degrees and pH 8.0. Recent kinetic studies on this system have led to the proposal that the cleavage of ATP bound to myosin is reversible.

Coupling of "high-energy" phosphate bonds to energy transductions.

Recent results suggest consideration of a new concept for oxidative phosphorylation in which a prime function of energy is to bring about release of ATP formed at the catalytic site by reversal of hydrolysis. Data with submitochondrial particles include properties of an uncoupler insensitive Pi=HOH exchange, a rapid reversible formation of bound ATP in presence of uncouplers, and predictable patterns of 32-Pi incorporation into ATP in rapid mixing experiments. ADP is confirmed as the primary Pi acceptor in mitochondrial ATP synthesis, but with chloroplasts ADP is also rapidly labeled.

Some chemical characteristics of dimethylsuberimidate and its effect on sarcoplasmic reticulum vesicles.

Sarcoplasmic reticulum vesicles treated with dimethylsuberimidate lose the capacity for ATP-promoted Ca2+ accumulation and show other properties indicative of leaky vesicles. As an aid to assessing whether this effect was caused by cross-linking or by hydrolysis products, characteristics of dimethylsuberimidate hydrolysis under incubation conditions used were measured. At pH 7.

The rapid labeling of adenosine triphosphate by 32P-labeled inorganic phosphate and the exchange of phosphate oxygens as related to conformational coupling in oxidative phosphorylation.

Evidence is presented that extends and amplifies the concept that in oxidative phosphorylation energy input serves to bring about release of ATP formed at a catalytic site by reversal of hydrolysis. The evidence with beef heart submitochondrial particles includes additional demonstration of uncoupler insensitive Pi leads to HOH exhchange, demonstration that this exchange is sensitive to the specific phosphorylation inhibitor, oligomycin, and demonstration that the small burst of uncoupler-insensitive ATP, rapidly labeled after addition of a tracer of 32Pi, behaves in a manner consistent with its participation as a membrane-bound intermediate in the Pi leads to HOH exchange. In addition, data are presented showing that addition of hexokinase plus glucose to submitochondrial particles in presence of ADP and Pi considerably lowers the Pi leads to HOH exchange but that further addition of cyanide or 2,4-dinitrophenol or both has little additional effect.

Isotopic probes of catalytic steps of myosin adenosine triphosphatase.

A new approach to the direct estimation of the value of the off constant for dissociation of ATP from myosin subfragment 1 (S1) has been developed. From measurements of the extremely slow rate of release of [32P] - ATP formed from 32P(i) by S1 catalysis and the amount of rapidly formed [32P] - ATP tightly bound to S1, the value of the off constant is approximately 2.8 X 10(-4) sec -1 at pH 7.