Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles.

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.

The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria.
Tightly bound ATP may also play an essential role.
In the accompanying, preceding paper we have described two methods which deplete soluble beef heart ATPase (F,) of "tightly bound" nucleotides while leaving the ATPase activity unchanged (1). These two methods were (a) trypsin treatment and (6)

Methods
Nucleotide Content of ETCH-The ATP and ADP content of washed ETPH was assayed fluorometrically following procedures described in the preceding paper (1). Samples of ETPH were prepared for analysis by washing four times in 0.25 M sucrose, 10 mM Tris/acetate, 1 mM EDTA, pH 7.5, centrifuging at 105,000 x g for 35 min between each wash to sediment the particles. The particles (0.5.ml samples) were extracted by addition of 10% (w/v) cold HClO, to a final concentration of 4% (w/v). About 5 to 6 mg of particles were assayed per experiment.
The denatured protein was removed by centrifugation and the supernatants assayed for ATP and ADP as described in the previous paper (1).
Gel Filtration of ETPH-Sephadex G-25 gel filtration of ETPH at 0" was done in columns (24 x 1.5 cm). The column buffers are described in Tables IV and V. ETPH was applied to the columns in samples of 0.5-ml volume containing 8 to 9 mg. The effluent particles were collected by centrifugation at 165,000 x g for 30 ruin and were resuspended in column buffer. Trypsin 7'reatment of ETCH-Samples of ETPH (1 mg/ml) in 0.25 M sucrose, 10 mM Tris/acetate, 1 mM dithiothreitol, 1 mM ATP, 4 mM MgCl,, pH 7.5, were incubated at room temperature with varying amounts of trypsin added as a 1 mg/ml of solution in 1 mM H,SO,. Soybean trypsin inhibitor was added to stop the reaction (5-fold excess by weight). Control experiments showed that trypsin had no effects at all if added after the soybean inhibitor.
ATPose Assays-ATPase activity was assayed calorimetrically as described in the preceeding paper (1).

Nucleotide
Content of ETPH--The content of tightly bound ATP and ADP in ETPH prepared here was slightly variable.
Values found are shown in Table I   and Energy-coupling Activity of gTPH-Trypsin treatment enhanced the ATPase activity of ETPH as shown in Fig. 1. These particles were centrifuged before assay, and the activity of treated particles remained oligomycin-sensitive.
At the same time trypsin treatment removed all ATP and ADP from the enzyme and eliminated energy coupling (Tables I and II). These results are therefore comparable to the results shown in the previous paper where the soluble F, was treated with trypsin, and loss of nucleotide and coupling activity from F, occurred. This together with the results of Slater et al. (5) showing that the tightly bound nucleotide in washed submitochondrial particles can be accounted for by F,-bound nucleotide suggests that trypsin, at low levels, first attacks F, in ETPH and causes loss of coupling. Control experiments were done to show that trypsin at the levels used did not affect NADH oxidase or succinate oxidase activity (Table III).
Effects of Sephadex G-25 Gel Filtration in High Ionic Strength Buffers on ATPase Activity, Nucleotide Content, and Coupling Activity of ETPH--In the preceding paper we showed that gel filtration of F, in a buffer of high ionic strength removed nucleotide and eliminated coupling activity of F,, while leaving ATPase activity unreduced and stable. We wished therefore to see if parallel experiments yielded similar results in ETPH.
First we passed ETPH through Sephadex G-25 columns in TE buffer (50 mM Tris/SO,, 1 mM EDTA, pH 8.0) or in TEK buffer (50 mM Tris/SO,, 1 mM EDTA, 60 mM K,SO,, pH 8.0) at either 4" or 20". Energy-coupling activity was completely lost in all cases. Subsequently, by trial and error, we alighted upon two low ionic strength buffer systems in which energy-

"Tightly
Bound" Nucleotides in ETPH coupling activity was retained after gel filtration of ETPH at 4" and these were (a) 0.25 M sucrose, 10 mM Tris/acetate, 4 mM MgCI,, 1 mM dithiothreitol, pH 7.5; and (b) 0.25 M sucrose, 10 mM Tris/acetate, 1 mM dithiothreitol, pH 7.5 or pH 8.0. The effect of ionic strength on the coupling activity and the nucleotide content of ETPH was then ascertained by adding K,SO, in increasing amounts to these buffers. The effects are documented in Tables IV and V. A correlation was noted between degree of removal of total tightly bound nucleotide and loss of energy-coupling activity. ATP could be removed completely without complete loss of coupling activity, and seemed to be removed more readily than ADP. In control experiments it was shown that addition of K&30, to the energy-coupling assay itself did not cause inhibition of NAD reduction.
Gel filtration of ETPH in these buffers increased ATPase activity markedly, and the activity remained very sensitive to inhibition by oligomycin (Table VI). DISCUSSION The results presented here extend the observations of the preceding paper (in which soluble F, was studied) to the membranous ETPH system. Clearly the two techniques which we found efficacious in removing tightly bound nucleotide from soluble F, are able to remove them from membrane-bound F,. Higher concentrations of trypsin were required in the mem- The gel filtration was done at 0" as described under "Methods." The column buffer was 0.25 M sucrose, 10 mM Tris/acetate, 1 mM dithiothreitol, 4 mM MgCl*, pH 7.5. "Tightly bound" ATP and ADP, and coupling activity were assayed in the particles as described under "Methods." Results are given as mean * S.D. with range and number of observations in parentheses.   gomycin-sensitive. Juntti et al. (4) have previously found that Finally, the fact that membrane-bound ATPase activity is trypsin treatment inactivated transhydrogenase activity and retained, indeed enhanced, in the nucleotide-depleted ETPH oxidative phosphorylation in "MgATP" submitochondrial par-preparations supports the suggestions made in the previous ticles, but had no effect on NADH oxidase, succinate oxidase, paper, namely that non-energy-linked ATPase activity and or oligomycin-sensitive ATPase. Similarly, some modifications membrane-binding properties of F, do not require tightly were required in the gel filtration system (higher ionic strength bound ATP or ADP. was required)

Sample
in order to remove all the tightly bound nucleotide, but again the F, remained membrane-bound,