THE JOURNAL OF BIOLOGICAL CHEMISTRY
`
`D 1988 by The American Society for Biochemistry and Molecular Biology, Inc
`Mitochondrial ATP Synthase
`OVEREXPRESSION IN ESCHERICHIA COLI OF A RAT LIVER p SUBUNIT PEPTIDE AND ITS
`INTERACTION WITH ADENINE NUCLEOTIDES*
`
`Vol. 263, No. 30, Issue of October 25, pp. 15694-15698,1988
`Printed in U. S. A.
`
`David N. Garboczi, Joanne H. Hullihen, and Peter L. Pedersen
`From the Laboratory for ~olecular and Cellular Bioe~rge~ics, Department of B ~ ~ g ~ c a l
`C ~ m ~
`The John? Hopkins School of Medicine, Baltimore, Maryland 21205
`
`~
`
`~
`
`,
`
`
`
`(Received for publication, June 3, 1988)
`
`EXPERIMENTAL PROCEDURES
`
`The C-terminal two-thirds of the rat liver ATP syn-
`thase. Many randomly produced mutations in the fl subunit
`thase subunit has
`been overexpressed and exported
`interfere with the assembly of the enzyme and so have been
`to the Escherichia coli periplasm under the direction
`relatively uninformative about the events of catalysis (9). The
`(phoA) promoter and
`of the alkaline phosphatase
`effects of other mutations which do not appear
`to affect
`leader peptide. The processed soluble protein contains
`structure frequently affect cooperativity between subunits (10,
`the 358 amino acids from glutamate 122 to the rat
`11).
`liver C-terminal serine 479, including all the regions
`In order to avoid some of the above difficulties in the
`that have been predicted by chemical and genetic mod-
`interpretation of mutation experiments on the intact ATP
`ification studies to be involved in nucleotide, Pi, and
`synthase complex, we have chosen to focus our studies both
`M S + binding.
`
`on the isolated @ subunit and peptide f r a ~ e n t s thereof. Here,
`Through a simple sequence of ~ r i s ~ D T A ~ y ~ z y m e
`we report the development of an overexpression system in
`treatment, osmotic lysis, and alkaline pH washes, the
`processed B subunit fragment can be prepared in >95%
`Escherichia coli producing the ATP synthase @ subunit frag-
`ment, C4.’ In addition, data are presented which demonstrate
`purity and at a yield of >20 mg/liter of‘ culture. It
`that this fragment, upon purification to near homogeneity,
`interacts with 2’(3’)-0-(2,4,6-trinitrophenyI) adeno-
`retains its capacity for tight nucleotide binding.
`sine 5’-triphosphate
`(TNP-ATP) which exhibits a
`strong enhancement of fluorescence upon binding. A
`similar enhancement is observed upon interaction with
`TNP-ADP. Enhancement observed with both TNP-
`nucleotides is markedly reduced by prior addition of
`either ATP or ADP and almost completely prevented
`by the ATP synthase inhibitor 7-chloro-4-nitrobenz-
`2-oxa-1,S-diazole. Both TNP-ATP and TNP-ADP bind
`at a stoichiometry of approximately 1 mol of nucleo-
`tide/mol of B subunit fragment. Under the same condi-
`tions, TNP-AMP does not exhibit a fluorescence en-
`hancement.
`This work demonstrates that, in the absence of inter-
`action with other ATP synthase subunits, the rat liver
`B subunit sequence from glutamate 122 to the C ter-
`minus exhibits no more than one readily detectable
`nucleotide binding domain. This success in producing
`a “functional” 0 subunit fragment has significance for
`the pursuit of genetic and physical studies focused on
`the structure and function of the rat liver ATP syn-
`thase 8 subunit.
`
`Materials
`The Klenow fragment of E. coli DNA ~ ~ y m e r a s e was obtained
`from United States Biochemicals. All other DNA modifying and
`restriction enzymes and phage M13mp19 DNA were obtained from
`New England Biolabs and were used according to the manufacturer’s
`instructions. Laboratory chemicals were from Sigma and J. T. Baker
`Chemical Co. The bicinchoninic acid protein assay was from Pierce
`Chemical Co. dNTPs were from Pharmacia LKB Biotechnology Inc.
`Agarose was from FMC Corp. and from Sigma. The BamHI-EcoRI
`adapter
`
`(5“GATCCTCGAG-3’
`3’-GAGCTCTTAA-5’)
`was from Amersham Corp. Acrylamide gel reagents were from Bio-
`Rad. NBDCI was from Sigma. TNP-nucleotides were purchased from
`Molecular Probes and their purities were confirmed by chromatog-
`raphy on polyethyleneimine-cellulose plates (Cel-300 PEI, Brink-
`mann Instruments) in a solvent system containing 2 M formic acid
`and 0.5 M LiC1. The expression plasmid pFOG402 and the bacterial
`the generous gifts of Dr. D. Shortle (Johns
`strain SE6004 were
`Hopkins School of Medicine).
`
`The catalytic sites of the mitochondrial ATP synthase are
`generally believed to reside on the @ subunits (for recent
`reviews, see Refs. 1-5). Evidence for this includes the findings
`that nucleotide analogs covalently label the p subunit (6, 7)
`and that some mutations in the @ subunit affect catalysis
`without seeming to alter other characteristics of the enzyme
`(8). The interpretation of the effects of mutations in the @
`subunit has been complicated by the complexity of interac-
`tions and cooperativity among the subunits of the ATP syn-
`* This work was supported by National Institutes of Health Grant
`CA 10951 to P. L. P. The costs of publication of this article were
`defrayed in part by the payment of page charges. This article must
`t.herefore be hereby marked “aduertisement” in accordance with 18
`U.S.C. Section 1734 solefy to indicate this fact.
`
`M e t ~ s
`Plasmid Construction-M13mpl9 containing the C4 @ subunit
`fragment was digested with EcoRI and Hind111 and the C4 fragment
`was purified from an agarose gel. pFOG402 was digested with BamHI
`‘ The abbreviations used are: C4, rat liver fl subunit fragment of
`mitochondrial ATP synthase containing the 358 amino acid residues
`extending from G1ulZ2 to the C-terminal Ser4“’ (bovine heart number-
`ing system); TNP-ATP, 2’(3’)-0-(2,4,6-trinitrophenyl)adenosine 5’-
`triphosphate; TNP-ADP, 2’(3’)-0-(2,4,6-trinitrophenyl)adenosine
`2’(3’)-~-(2,~,6-t~nitrophenyI)adenosine
`5”~phosphate; TNP-A~P,
`5’-monophosphate; MOPS, 3-(N-morpholino)propanesulfonic acid;
`Tricine, N-tris(hydroxymethy1)methylglycine; PMSF, phenylmeth-
`ylsulfonyl fluoride; CAPS, 3-cyclohexylamino-1-propanesulfonic
`acid NBDCI, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole; AMP-PNP, ad-
`enyl-5”yI imidodiphosphate.
`
`15694
`
`KASHIV EXHIBIT 1071
`IPR2019-00791
`
`Page 1
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`

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`15695
`
`pFOG402
`
`adapter
`
`RESULTS AND DISCUSSION
`Modification of the p Subunit Fragment C4 for Expression-
`Recently this laboratory described the screening of a rat liver
`cDNA library with anti-F1 antiserum resulting in the isolation
`of cDNAs spanning all but seven amino acids of the rat liver
`ATP synthase p subunit (13). During attempts to overexpress
`the
`the almost full-length /3 subunit, it was inserted into
`alkaline phosphatase expression vector pFOG402. This vector
`was chosen because of the reported strength of the inducible
`alkaline phosphatase promoter (14) and because overproduced
`proteins from this vector may be exported to the periplasmic
`space (15). When bacteria harboring the expression plasmid
`containing the p subunit cDNA were grown in low phosphate
`medium, a high level of protein was produced of a size con-
`sistent with its being the @ subunit plus the alkaline phospha-
`tase leader peptide (precursor (3). At the same time, very little
`protein of a size consistent with its being the p subunit after
`removal of the leader peptide (processed p) was seen (results
`
`Expression and Nucleotide Binding of Rat Liver Fl-p Peptide
`7.4, aliquots of the C4 solution were used for fluorescence measure-
`at the single BamHI site at the C terminus of the alkaline phosphatase
`ments. This was performed in a 4-ml quartz cuvette (Precision Cell
`leader peptide (Fig. 1). The BamHI ends were made blunt-ended with
`Inc.) in 2 ml of
`50 mM Tris-SO4, 0.5 mM EDTA, pH 7.5, and
`Klenow fragment in the presence of dNTPs to prevent the ligation
`concentrations of TNP-nucleotides and C4 as indicated in the legend
`of the adapter described below to the cohesive BamHI ends of the
`to Fig. 4. ATP and ADP were prepared as 100 mM, pH 7.4, stock
`vector. HindIII was then used to cut at the single HindIII site within
`solutions in water. NBDCl was prepared as a 100 mM stock solution
`the Staphylococcal nuclease gene, thereby creating a 325-base pair
`in 50% ethanol, 50% water. An Amino SPF-125 spectrofluorometer,
`fragment and the vector fragment. The larger vector fragment was
`set at an excitation wavelength of 405 nm (slit width of 1) and an
`purified from an agarose gel.
`emission wavelength of 520 nm (slit width of 2), was used for these
`The C4 fragment and the pFOG402 vector fragment were mixed
`studies.
`at a molar ratio of 1:1 in a ligation reaction with T4 DNA ligase and
`(1.5 mm
`Electrophoresis and Protein Assay-Polyacrylamide gels
`incubated at 15 "C for 3 h. A BamHI-EcoRI adapter at 100-fold molar
`thick, 15% acrylamide), using the Laemmli buffers, were used for the
`excess and additional T4 DNA ligase were then added to the reaction
`analysis of proteins. The bicinchoninic protein assay was used to
`and the incubation continued at 4 "C for 13 h. After two ethanol
`determine protein concentrations using 40,000 as the computed mo-
`precipitations, the remaining cohesive ends were filled in with Klenow
`lecular weight of C4 and using bovine serum albumin as the standard.
`fragment and dNTPs for 30 min at 23 "C. Additional T4 DNA ligase
`was added, and the incubation resumed at 4 "C for 36 h.
`An aliquot of the ligation was used to transform strain DH1 to
`ampicillin resistance. Plasmids were sized on agarose gels after DNA
`purification by alkaline-SDS lysis. Bacteria containing appropriately
`sized plasmids were grown on low phosphate medium (described
`below) at 37 "C. Aliquots (25 pl) of cultures grown on low phosphate
`medium were heated in SDS/mercaptoethanol and analyzed on poly-
`acrylamide gels for the presence of Coomassie Blue-stained p subunit
`peptides that were of the expected size and that reacted with rat liver
`/3 subunit antisera (not shown).
`Expression and Purification of C4"To obtain better processing of
`the leader peptide, plasmid DNA purified by the alkaline-SDS method
`was used to transform E. coli strain SE6004 to ampicillin resistance.
`SE6004 contains a mutation isolated by its ability to suppress some
`kinds of mutations that impede leader peptide processing (12).
`SE6004 cells containing the C4 expression plasmid were grown to
`phosphate-limited stationary phase in LB medium (10 g of Bacto
`Tryptone + 5 g of Bacto Yeast extract + 10 g of NaCl + H 2 0 to 1
`liter) containing 50 pg/ml ampicillin. The cells were then diluted 25-
`fold into complete MOPS medium which, for 1 liter, consists of 200
`ml of solution M (42 g of MOPS, 4 g of Tricine, 14.6 g of NaC1, 8 g
`of KOH, 2.55 g of NH4CI, and H20 to 1 liter), 2 ml of solution 0 (a
`500-ml solution containing 26.8 g of MgC12. 6H20, HZO, and 10 ml of
`a 100-ml solution containing 8 ml of concentrated HC1,5 g of FeClz.
`4H20, 184 mg of CaC12.2Hz0, 64 mg of H3B03, 40 mg of MnCl2.
`4H20, 18 mg of CoClz. 6H20, 4 mg of CuC12. 2H20, 340 mg of ZnC12,
`605 mg of NaaMoc. 2H20, and HzO), 0.1 ml of 1 M KH2P04, 1.0 ml of
`0.276 M K2SO4, 20 ml of 20% glucose, 1 ml of 0.1% thiamine, 40 ml
`of 3.75% vitamin-free casein hydrolysate, H20, and 50 pg/ml ampi-
`cillin. After a 10-14-h incubation at 30 "C, the cells were either
`analyzed on polyacrylamide gels or used to purify the C4 protein.
`Two liters of cells were induced for purification of C4. Cells were
`pelleted at 4000 rpm for 10 min in a Sorvall GSA rotor and resus-
`pended in 400 ml of 200 mM Tris-C1, pH 8.0, 20% sucrose at 23 "C.
`NaEDTA (1 mM), lysozyme (60 pglml), and PMSF (0.1 mM) were
`added immediately and mixed. After 5 min, this suspension was
`diluted into 800 ml of 1 mM NaEDTA and 0.1 mM PMSF and
`incubated with stirring for 20 min at 23 "C. Cells were pelleted at
`7500 rpm for 20 min in the GSA rotor. Pellets were resuspended in a
`minimal quantity (20 ml) of cold 200 mM Tris-C1, pH 8.0,20% sucrose,
`0.1 mM PMSF and immediately diluted into 250 ml of cold 1 mM
`NaEDTA and 0.1 mM PMSF with stirring. After 5 min the suspension
`was brought to 5 mM MgClz, 3 pg/ml DNase, and was briefly warmed
`to aid DNase digestion. After the viscosity lessened, the suspension
`was centrifuged in a Sorvall SS-34 rotor at 15,000 rpm for 20 min.
`Pellets were washed two times by resuspension in 60 ml of 30 mM
`Tris-C1, pH 8.0, 1 M NaC1, 1 mM NaEDTA, 0.1 mM PMSF and
`centrifuged in the SS-34 rotor at 15,000 rpm for 20 min. Pellets were
`then washed two times by resuspension in 10 ml of 30 mM CAPS, pH
`10.6, 1 mM NaEDTA, 0.1 mM PMSF and pelleted at 48,000 rpm in
`the Beckman T65 rotor for 30 min. The bulk of the processed C4
`appeared in the supernatant of the second high pH wash.
`Amino Acid Sequence of C4"The sample for sequencing analysis
`was prepared by dialyzing 1 ml (5 mg) of C4 overnight against two
`changes of distilled water (4 and 1 liter, respectively). The resultant
`precipitated C4 was washed once in 1 ml of distilled water and then
`redissolved in 1.1 ml of 30% acetonitrile, 10% acetic acid in water.
`
`
` An aliquot (5 p ~ ,20 pg of C4) was lyophilized, redissolved in 40%
`acetonitrile, 5% acetic acid in water, and subjected to amino acid
`sequence analysis on an Applied Biosystems Model 470A amino acid
`sequencer in the University Protein/Peptide Facility.
`Interaction of C4 with TNP-nucleotides-After dialysis of the sec-
`ond high pH supernatant against 100 volumes of 2 mM Tris-C1, pH
`
`u
`u
`
`E: B: EcoRl BamHl
`
`H: Hlndlll
`
`Klenow, dNTPs
`llgase
`
`FIG. 1. Construction of the FI-B fragment C4 expression
`plasmid. Shown are the steps performed to ligate the C4 cDNA and
`adapter oligonucleotide into the expression plasmid. Part of the
`Staphylococcal nuclease coding region was removed in the first step
`and replaced by the C4 cDNA (EcoRI-Hind111 fragment). (See "Meth-
`ods" for detailed description.)
`
`Page 2
`
`

`

`15696
`
`1
`
`2
`
`> > -
`
`45
`
`- 21
`
`- 14
`
`-”
`-mas===,
`
`43 -
`
`25 -
`
`14-
`
`Expression and Nucleotide Binding of Rat Liver K-/3 Peptide
`
`
`
`1 2 3 4 5 6 1 2 3
`3
`4
`
`-
`” - 92
`CE “
`-
`I-=
`66
`66 -
`
`-
`
`L
`
`<-
`
`I==
`
`FIG. 2. Processed F1-j3 fragment C4 is extractable from the
`periplasm. Lane I , supernatant containing processed C4 (lower
`arrow at left) obtained by treatment of the cells from 100 pl of
`bacterial culture with lysozyme and 1 M Tris base, pH 10.5. Lunes 2
`and 3,50 pl of bacterial culture showing unprocessed C4 (upper arrow
`at left) and processed C4 (lower arrow at left) to differ in molecular
`weight, consistent with the leader peptide still being part of the
`unprocessed C4. Lane 4, standard proteins visible from top to bottom:
`phosphorylase b (92,000), bovine serum albumin (66,200), F1-ATPase
`01 subunit (55,000), F1-ATPase p subunit (51,000), ovalbumin (45,000),
`F,-ATPase y subunit (33,000), carbonic anhydrase (31,000), and
`trypsin inhibitor (21,000).
`
`not shown). This result indicated that the /3 subunit precursor
`protein was not being processed by leader peptidase or by
`other bacterial proteases.
`One possibility for incomplete processing is the lack of
`protease recognition of the amino acid sequence at the pre-
`dicted cleavage site. As the Staphylococcal nuclease protein
`produced from pFOG402 is processed well in E. coli, we added
`an oligonucleotide adapter encoding additional N-terminal
`amino acids to the /3 subunit cDNA in order to mimic those
`of the leader peptidase cleavage site in the Staphylococcal
`nuclease construct. As shown in Fig. 1, the same oligonucle-
`otide adapter was added to a shorter /3 subunit cDNA (C4)
`that encodes the C-terminal two-thirds of the subunit. These
`amino acids in the nuclease were Arg-Ile-Asp-Pro- where the
`peptide bond between the arginine residue and the preceding
`amino acid of the leader peptide is cleaved by leader peptidase,
`making arginine the N-terminal residue of the processed
`protein (14). After adding an adapter oligonucleotide to each
`plasmid, the predicted N-terminal amino acid sequence of the
`processed p subunit and of the processed C4 is Arg-Ile-Asp-
`Pro-Arg. An additional arginine residue was included in the
`modified sequence through the use of this particular adapter.
`This modification of the
`subunit plasmid improved the
`expression of the apparently processed @ subunit, allowing
`the accumulation of a significant amount (5% of precursor)
`of processed protein (not shown). Work is in progress to
`purify the p subunit from cells containing this modified plas-
`mid.
`In contrast to the full-length p subunit, however, C4 was
`highly expressed and accumulated to a level that is approxi-
`mately 40% of the precursor C4 (Fig. 2). As seen in Fig. 2,
`
`A
`B -
`FIG. 3. Steps in the purification of C4. A, lane I, soluble extract
`(25 pl) of bacteria lysate; lane 2, supernatant (10 pl) from 1 M NaCl
`wash of lysed bacteria; lane 3, supernatant (5 pl) from 30 mM Tris-
`C1, pH 8.0, wash; lane 4, 13 pg of high pH wash supernatant; lane 5,
`50 pg of high pH wash supernatant; lune 6, 25-p1 aliquot of bacterial
`culture. B, lunes 1, 2, and 3 are 1.25, 2.5, and 5 pg of protein,
`respectively, obtained from the second high pH wash. The C4 protein
`shown in B is a separate preparation from A. See “Methods” and
`“Results and Discussion” for details.
`
`precursor and processed C4 accounted for a large fraction of
`bacterial cell protein; an aliquot (50 111) of the cell culture was
`loaded in lanes 2 and 3. We obtained an additional increase
`in the yield of processed C4 (to 50% of precursor) by incubat-
`ing the induced cultures at 30 “C instead of 37 “C.
`Purification of the Rat Liver p Subunit Fragment C4”Since
`C4 was expressed fused to the alkaline phosphatase leader
`peptide, we looked for the appearance of C4 in the periplasm.
`Treatment of the cells with osmotic shock, known to release
`some proteins of the periplasmic space (16), did not release
`any C4 to the supernatant. We then tried resuspending the
`cells in 1 M Tris base, 2.5 mM NaEDTA, pH 10.5, a technique
`that releases recombinant Staphylococcal nuclease produced
`from pFOG402 (15, 17). This also did not release C4. Treat-
`ment of the cells with lysozyme followed by treatment with 1
`M Tris did release C4 (Fig. 2). The ability to release C4 from
`the periplasm is one indication that C4 is being exported.
`Since leader peptidase is located in the periplasmic space (18),
`the existence of processing can be taken as an indication that
`the protein is being exported (19). As C4 was being signifi-
`cantly processed and appears to be extractable from the
`periplasm, it was likely being exported, which would simplify
`its purification. The method described above, although effec-
`tive at releasing C4, produced unacceptable cell lysis.
`Since C4 appeared to be tightly associated with membranes,
`washed bacterial membranes were prepared and C4 was spe-
`cifically released by alkaline pH. This technique consisted of
`Tris/EDTA/lysozyme.treatment, osmotic lysis, and high salt
`
`Page 3
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`

`

`Expression and Nucleotide Binding
`
`15697
`
`60 ] A
`
`0
`
`2
`
`4
`
`8
`6
`
`10
`12
`TNP-ATP (/A)
`
`14
`
`16
`
`50 --
`
`40 --
`30 -_
`
`6
`
`
`
`Fl-p Peptide
`of Rat Liver
`ligation of the oligonucleotide adapter.
`isolated p
`Interaction of C4 with TNP-nucleotides-The
`subunit is known to bind one (20) and possibly two ATP or
`ADP molecule(s) per molecule of subunit (21-23). To deter-
`mine if the overproduced C4 would also bind nucleotides, the
`interaction of TNP-nucleotides and C4 was studied by meas-
`uring the enhancement of fluorescence that occurs upon the
`binding of TNP-nucleotides. When 0-15 @M TNP-ATP or
`TNP-ADP was added to a cuvette (see “Methods”) containing
`6 p~ C4, the binding curves displayed in Fig. 4 were generated.
`TNP-ATP and TNP-ADP both exhibited a marked enhance-
`ment of fluorescence over the fluorescence of each nucleotide
`in the absence of C4. The apparent linearity of the initial
`portion of the TNP-ATP and the TNP-ADP curves indicates
`a high affinity ( K d 5 5 PM) of the nucleotide analogs for the
`C4 protein. TNP-AMP did not exhibit an enhancement
`of
`fluorescence. Stoichiometries (i.e. mol of TNP-nucleotide/
`mol of C4) estimated, as indicated in the
`legend to Fig. 4,
`were near 1 for both TNP-ATP and TNP-ADP and did not
`exceed 1.3 in several different experiments.
`The addition of 0.5 mM ATP to the cuvette before titration
`with TNP-ATP or TNP-ADP markedly reduced the amount
`of fluorescence enhancement obtained (Fig. 4). The addition
`of 0.5 mM ADP also decreased the fluorescence enhancement
`of both TNP-nucleotides to a similar degree. These results
`indicate that a single nucleotide binding site is present on C4
`which can interact with both di- and triphosphate adenine
`nucleotides and analogs thereof. Consistent with this conclu-
`sion, TNP-ADP failed to further enhance the maximal fluo-
`rescence observed at 15 PM TNP-ATP, and TNP-ATP failed
`to further enhance the maximal fluorescence observed at 15
`FM TNP-ADP (data not shown).
`NBDCl has been shown to be a covalent inhibitor of various
`F1-ATPases (24, 25). In rat liver F,-ATPase, the binding of
`NBDCl prevents the binding of AMP-PNP to the enzyme
`(26). When NBDCl is added to a solution of C4, an enhance-
`ment of fluorescence over that seen with NBDCl alone is
`seen. In Fig. 4 is shown the effect of adding TNP-nucleotide
`to the NBDC1-modified C4 in the assay cuvette. NBDCl
`completely prevents the enhancement of fluorescence indic-
`ative of TNP-nucleotide binding. If, however, the NBDCl is
`added to C4 that is already in the presence of 15 KM TNP-
`nucleotide, no change in the
`level of fluorescence is seen,
`
`indicating that the binding of TNP-nucleotide is not disrupted
`and pH washes (detailed under “Methods”) and produced C4
`and that the binding of NBDCl to C4 is prevented by the
`in a relatively high state of purity. In Fig. 3A an acrylamide
`prior binding of the nucleotide.
`gel of purification fractions and of C4 prepared with a 30 mM
`That the overexpressed C4 protein interacts with TNP-
`Tris-C1, pH 8.0 wash instead of a second high pH wash is
`ATP and TNP-ADP signifies that the bacterially made rat
`shown. Note in Fig. 3A, lune 2, that treatment of the mem-
`liver protein possesses two of the functional characteristics of
`branes with 1 M NaCl releases several
`abundant proteins
`the p subunit. Upon interaction with the TNP-nucleotides,
`without releasing C4. In Fig. 3B, C4 was prepared with two
`the observed enhancement of fluorescence is similar to that
`high pH washes and is apparently homogeneous with minor
`reported with bovine heart F1-ATPase (27) and with a syn-
`bands becoming visible only at high protein loading. The yield
`thetic 50-amino acid peptide, pp50 (28), modeled on a putative
`of processed C4 is 20-25 mg/liter of induced cells.
`nucleotide binding site on the
`/3 subunit. Unlike the simplified
`Significantly, amino acid sequencing analysis of purified C4
`sequence of pp50, the C4 sequence contains all of the regions
`confirmed that the C4 precursor was cleaved at the predicted
`
`that are thought to be important for ATP, ADP, Pi, and Mg2+
`processing site. The N-terminal sequence of the first 25 amino
`binding and its study should complement the study
`of the 50-
`acids of the processed C4, determined as indicated under
`amino acid pp50 peptide. The fact that the nucleotide binding
`“Methods,” is shown below.
`site of the p subunit can now be studied not only in F1-
`Arg-Ile-Asp-Pro-Arg-Glu-Phe-Ile-Glu-Met-Ser-Val-Glu-
`ATPase, but in
`two much simpler proteins, C4 and pp50,
`Gln-Glu-Ile-Leu-Val-Thr-Gly-Ile-Lys-Val-Val-Asp
`should lead to detailed insight into the molecular nature of
`nucleotide binding to the ATP synthase.
`The underlined amino acids correspond to those previously
`liver p
`reported by us from cDNA sequencing
`of the rat
`Acknowledgments-We are especially grateful to Dr. David Shortle
`subunit (13) and
`commence with G1u’22. The amino acids
`of this department for his constant advice and encouragement while
`preceding the p subunit sequence are those predicted from the
`this work was in progress. We are indebted also to Dr. P. Shenbaga-
`
`
`10
`8 12
`
`TNP-ADP
`(/AM)
`FIG. 4. Interaction of C4 with TNP-nucleotides. Fluores-
`cence measurements were carried out as described under “Methods.”
`The concentration of C4 was held constant at 6 NM and the indicated
`concentrations of the specified TNP-nucleotide in A and B were
`added. The effects of the addition of 0.5 mM ATP and of 0.5 mM
`NBDCl before the TNP-nucleotide is shown in the lower plots in each
`panel and is discussed in the text. The relative fluorescence enhance-
`ment plotted is the fluorescence (in arbitrary units) of the TNP-
`nucleotide. C4 complex minus the fluorescence of the TNP-nucleotide
`alone. Stoichiometries (ie. mol of TNP-nucleotide/mol of C4) were
`estimated by extrapolation from the initial part of the titration curves,
`where it is assumed that all substrate is bound, to the maximal
`fluorescent change.
`
`0.5 mM ATP
`
`&ALA 0.5 rnM NBDCl
`
`14
`
`16
`
`2
`
`
`
`
`
`0 4
`
`--------”
`”
`
`_“”_”
`
`Page 4
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`Expression and Nucleotide Binding
`15698
`murthi of the University Protein/Peptide Facility for carrying out
`the amino acid sequence analysis.
`
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