©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Rat Choline Acetyltransferase by Limited Proteolysis (*)

(Received for publication, December 8, 1994; and in revised form, June 8, 1995)

Donghai Wu (§) S. Newaz Ahmed W. Lian Louis B. Hersh

From the Department of Biochemistry, University of Kentucky Lexington, Kentucky 40536-0084

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the past, purification of choline acetyltransferase (ChAT, EC 2.3.1.6), the enzyme responsible for the biosynthesis of the neurotransmitter acetylcholine, has yielded fragmented species of the enzyme. The nature and possible function of these forms of ChAT are not well understood. Using a bacterial expression system, recombinant rat ChAT in its active form has been purified to homogeneity. The purified enzyme was found to be activated to >25-fold when assayed at low ionic strength and >5-fold when assayed at high ionic strength by limited proteolysis with either trypsin or chymotrypsin, but not with proteinase K. The activated ChAT shows an increased K for both substrates, diminished sensitivity to salt activation and a pH optimum that is shifted approximately 1 pH unit. On a denaturing SDS-polyacrylamide gel, the activated ChAT is composed of three to four polypeptides; however, it migrates as an intact 68-kDa protein species on gel filtration. In order to delineate the site of cleavage by proteolysis, the newly generated fragments have been subjected to N-terminal sequencing. By comparing cleavage sites between trypsin and chymotrypsin, the putative activation sites were identified.


INTRODUCTION

Choline acetyltransferase (ChAT, (^1)EC 2.3.1.6) catalyzes the reversible transfer of an acetyl group between acetyl coenzyme A and choline, resulting in the synthesis of the neurotransmitter acetylcholine(1) . Since its discovery more than 50 years ago(2) , ChAT has been extensively studied, but little is known about structure/function relationships for this molecule as a catalyst. Initially, the kinetic mechanism catalyzed by this enzyme was postulated as an ordered bireactant or ping-pong mechanism(3) . Now it is generally accepted that this is predominantly an ordered sequential Theorell-Chance mechanism with acetyl coenzyme A as the leading substrate, although a minor component of a random sequential pathway does occur(4) . The reaction is thought to proceed via general base catalysis by an imidazole residue enhancing the nucleophilicity of the hydroxy group of choline to attack the thioester bond in the forward reaction(5) . The presence of an active site histidine was first postulated by White and Cavallito(6) . Recently, this active site histidine has been identified by site-directed mutagenesis as His-426 in Drosophila ChAT(7) .

Over the years multiple forms of choline acetyltransferase, which vary in charge and size, were observed using either column chromatography or electrophoretic methods(8, 9, 10) . However, the nature and physiological function of these variants remain unclear. Multiple charge forms of ChAT have been observed in all sources from which ChAT has been purified(8, 9, 10) . They do not represent distinct gene isoforms since the ChAT gene is a single copy in the genome(11) , nor do they represent alternative splicing products since a single transcript has the capacity to generate them(12) . Instead, multiple charge forms have been attributed to the possible association with other proteins(13) , to deamidation(14) , or to phosphorylation(15) . On the other hand, the size variants of ChAT may simply reflect artifacts generated during purification since they appeared to be somewhat variable depending on which purification scheme was used. For example, in the case of bovine brain ChAT, molecular sizes of 100-120 kDa(6, 8) , 72-76 kDa(13) , 65-70 kDa (16, 17) and six subunits of 14.7 kDa (18) have all been reported. Hersh et al.(19) attempted to address the nature of size and charge heterogeneity of ChAT from bovine brain and found that the 68-kDa form can be converted to a 63-kDa form by exogenously added Staphylococcus aureas protease, but not by autolysis in the extracts. However, only a 73-kDa form was observed when purification was carried out in the presence of a variety of proteolytic inhibitors(19) . Recently, Benecke et al.(20) reported the presence of 45- and 30-kDa forms, but not the intact 68-kDa form in partial purified ChAT samples. A 68-kDa form was, however, obtained in immunoaffinity-purified ChAT preparations(20) , suggesting that multiple sizes of ChAT were caused by proteolysis. In the case of Drosophila, cleavage of ChAT from 68 to 57 kDa and 13 kDa occurred with the wild-type enzyme, but not with two temperature-sensitive mutants(21) , indicating that proteolytic processing may be important for ChAT function. Thus, these studies clearly demonstrated the presence of proteolysis of ChAT, although the consequence and/or effects on catalysis were not investigated.

In order to address the nature of different size variants of ChAT and to better understand the process and effects proteolysis has on ChAT activity, limited proteolysis was carried out with known proteinases. In this report, we describe the activation of recombinant rat ChAT by limited proteolysis and the characterization of proteolytically activated enzyme.


EXPERIMENTAL PROCEDURES

Plasmid Constructs

A rat ChAT cDNA clone, previously isolated in two fragments(11) , corresponding to nucleotides 181-1604 and 1153-2153(22) , was used to link a polyhistidine to the N terminus of recombinant rat ChAT. An overlapping NdeI site was used to first join the two cloned fragments. Two additional oligonucleotides were then used to introduce a unique BglII site and a Factor Xa site at the 5` end, such that the rat ChAT cDNA could be fused inframe with the polyhistidine in the expression plasmid pQE9 (Qiagen), with the Factor Xa site behind it. The final expression plasmid was constructed by three-fragment ligation with BamHI- and SalI-digested pQE9, BglII- and KpnI-digested 5` fragment, and KpnI- and XhoI-digested 3` fragment. The construct was identified by colony hybridization, and the polymerase chain reaction-generated fragments were checked by dideoxy sequencing(23) .

Expression and Purification Of Recombinant ChAT

To express the recombinant rat ChAT, Escherichia coli SG12036 containing the above expression plasmid was grown at 22 °C for 4 days in LB media. Cells were harvested by centrifugation and frozen at -80 °C until further use. The recombinant enzyme was purified in a two-step procedure that involved binding of the introduced N-terminal polyhistidine to immobilized nickel, followed by dye binding chromatography. All purification steps were performed at 4 °C. 1 g of frozen cells was resuspended in 10 ml of 20 mM sodium phosphate buffer, pH 7.6, containing 1 mM phenylmethylsulfonyl fluoride, 2 µM benzamidine, 10 mM MgSO(4), and 2 µg/ml DNase I. Cells were broken by passage twice through a French pressure cell at 9,000-16,000 p.s.i. The homogenate was centrifuged at 39,000 g for 15 min, and the supernatant was applied directly to a 3-ml column of nickel-tritrilotriacetic acid-agarose (Qiagen) equilibrated with 20 mM sodium phosphate buffer, pH 7.6. The column was washed successively with 50 ml of the phosphate buffer containing (a) 0.1 M NaCl, (b) 1.0 M NaCl, (c) 2.0 M NaCl, (d) 1.0 M NaCl + 0.1% Triton X-100, (e) buffer alone, and finally (f) buffer + 5 mM imidazole, pH 7.4. The enzyme was eluded with a concave gradient of 5-250 mM imidazole, pH 7.6. The active fractions were pooled, diluted 1/10 with 20 mM sodium phosphate buffer, pH 7.6, and applied directly to a 5-ml column of blue agarose (Amicon), previously equilibrated with 20 mM sodium phosphate buffer, pH 7.6. After washing the column with 10 column volumes of equilibration buffer, the enzyme was eluted with a linear gradient to 2 M sodium chloride. The enzyme, which eluted at approximately 0.5 M NaCl, was concentrated/dialyzed in an Centricon 30 concentrator (Amicon). A summary of the purification scheme is given in Table 1. Analysis of the purified enzyme by SDS-PAGE revealed a single band. The addition of the polyhistidine to the enzyme had no effect on its kinetic properties as demonstrated by its removal with Factor Xa. (^2)



ChAT Activity Assay

Enzymatic activity was determined by either the radioactive assay of Fonnum (24) or the fluorometric assay of Hersh et al.(25) . The reaction mixture for the radioactive assay routinely contained 50 mM sodium phosphate buffer, pH 7.4, 100 mM sodium chloride, 0.5 mM EDTA, 15 µM eserine, 0.2 mM [^3H]CoASHAc (specific activity = 2 10^7 cpm/µmol) and 10 mM choline in a total volume of 50 µl. The reaction was started by the addition of enzyme and incubated at 37 °C for 10 min, after which time an aliquot was removed and the formation of [^3H]acetylcholine was determined as described previously (24) .

The fluorometric assay followed the reverse reaction in which acetyl-CoA formation was measured in a coupled assay using citrate synthase and malate dehydrogenase. Reaction mixtures of 0.4 ml contained 50 mM sodium phosphate buffer, pH 7.4, 100 mM sodium chloride, 0.25 mM NAD, 0.5 mML-malate, 0.1 mMDL-dithiothreitol, 1.0 unit of pig heart citrate synthase, 5 units of pig heart malate dehydrogenase, and 10 mM acetylcholine chloride and 200 µM CoA. The reaction was initiated by the addition of acetylcholine chloride, and NADH formation was monitored over a 3-5 min time period using a Ratio-2 fluorometer equipped with a strip chart recorder. An excitation wavelength of 340 nm and an emission wavelength of 460 nm were used.

Limited Proteolysis of ChAT

Stock solutions of trypsin, chymotrypsin, and soybean trypsin inhibitor were prepared daily. Limited proteolysis of ChAT was routinely carried out at 22 °C in 10 mM sodium phosphate, pH 7.6, with one mg/ml of ChAT and a proteinase to ChAT ratio of 1:10. The reaction with trypsin was terminated by the addition of 2 mg of soybean trypsin inhibitor/mg of trypsin. In those experiments, where physical properties such as molecular size determination, N-terminal sequencing, pH activity studies, and kinetic measurements were investigated, the proteinase-treated ChAT was separated from proteinase by gel filtration and concentrated/dialyzed for further studies.

Protein Determination, Gel Filtration, and Electrophoresis

Protein concentrations were determined by the BCA protein assay (Pierce), using bovine serum albumin as the standard. Gel filtration was performed according to the manufacturer's recommended protocol using a Superdex-75 column from Pharmacia Biotech Inc. Discontinuous SDS-PAGE was performed by the method of Lamelli (26) using a 10% acrylamide gel run at a constant current of 20-40 mA/gel. Gels were silver-stained by the method of Bloom et al.(27) .

Kinetic Properties of Recombinant ChAT

Measurement of the initial rates of enzymatic acetyl group transfer utilized the conditions described for the fluorometric enzyme assay with substrate concentration varied in the range of 0.3-5.0 the K. The K for acetylcholine was measured at a saturating concentration of CoASH (200 µM), while the K for CoA was measured at a saturating concentration of acetylcholine (20 mM). Data from kinetic experiments were fit to the weighted least squares kinetic programs of Cleland(28) .

The pH-Activity Studies

The pH-activity profile for the enzyme was obtained by assaying the enzyme in 5 mM sodium phosphate buffer with or without added NaCl (250 mM) at the desired pH. Assays were conducted at 0.2 mM acetyl-CoA and 5 mM choline using the radioactive assay(24) .

N-terminal Sequence of Trypsin and Chymotrypsin-activated ChAT

100 µg of ChAT proteolytically activated either by trypsin or chymotrypsin were denatured and separated by SDS-PAGE on a 15% polyacrylamide gel as described above and transferred on to a polyvinylidine difluoride membrane. The membrane was first stained with 0.5% Ponceau S, and after destaining in 5% acetic acid, each band was excised and subjected to amino acid sequence analysis with a model 477A Applied Biosystems protein sequenator equipped with an on-line phenylthiohydantoin analyzer by the University of Kentucky Molecular Structure Core. Similarly, 50 µg of ChAT, proteolytically activated either by trypsin or chymotrypsin, were directly applied onto polyvinylidine difluoride membranes and subjected to N-terminal sequence determination.


RESULTS

Activation of Rat Choline Acetyltransferase By Trypsin

Highly purified recombinant rat choline acetyltransferase was incubated in the presence of 0.5 mg/ml of trypsin in 10 mM phosphate buffer, pH 7.6. Fig. 1shows that incubation of ChAT with trypsin produces a time-dependent 5.5-fold increase in enzyme activity. Incubation with increasing concentrations of trypsin results in a similar 5-6-fold increase in ChAT activity when assayed in the presence of 100 mM sodium chloride. Maximal activation was achieved at a trypsin concentration of 100 µg/ml for a 30-min incubation period. Decreasing the trypsin concentration below 100 µg/ml increased the time required to achieve maximum activation. However, increasing the trypsin concentration above 100 µg/ml was ineffective and actually decreased the maximal activation achieved, presumably due to secondary cleavages. In order to minimize this effect, 100 µg/ml trypsin was chosen with a 30-min incubation time at 22 °C as the standard activation condition for subsequent experiments.


Figure 1: Effect of trypsin treatment on the activity of recombinant rat choline acetyltransferase. Trypsin digestion (bullet) was carried out at 22 °C in 10 mM sodium phosphate, pH 7.6, with 1 mg/ml of ChAT and 1 µg of trypsin in a reaction volume of 10 µl. As a control (), enzyme was incubated under the same conditions in the absence of trypsin. The reaction was stop by the addition of 2 mg of soybean trypsin inhibitor/mg of trypsin, and enzymatic activity was determined by the standard fluorometric assay after a 200-fold dilution of the reaction mix.



Molecular Size of the Activated Choline Acetyltransferase

The molecular size of ChAT after treatment with trypsin was compared with that of the native enzyme by molecular sieve chromatography. As shown in Fig. 2, trypsin-activated ChAT migrated at the same position as native ChAT, indicating first, that trypsin activation does not result from the formation of a stable complex with ChAT, and second that trypsin does not remove a large inhibitory domain of ChAT. In order to find out where cleavage had occurred, trypsin-activated ChAT was first separated from trypsin by gel filtration, denatured under reducing conditions, and analyzed by SDS-PAGE. Fig. 3shows one band of 68 kDa for native ChAT prior to trypsin incubation and at least three distinct bands of 31 kDa (Ta), 21 kDa (Tb), and 14 kDa (Tc1, Tc2, and Tc3) after trypsin treatment, indicating nicking and/or gapping was caused by proteinase treatment.


Figure 2: Gel filtration of rat recombinant choline acetyltransferase before and after incubation with trypsin. Opencircles are data for native choline acetyltransferase, and closedcircles are data for trypsin-treated choline acetyltransferase chromatographed on a Superdex 75 column at flow rate of 0.5 ml/min using 10 mM sodium phosphate buffer, pH 7.6, containing 100 mM sodium chloride.




Figure 3: SDS-PAGE analysis of recombinant choline acetyltransferase before and after limited proteolysis with trypsin. Native recombinant rat ChAT (5 µg; lane2) and trypsin-activated ChAT (5 µg; lane3) were analyzed on a 10% SDS-PAGE. Proteins were visualized by silver staining. The resultant tryptic peptides are labeled Ta, Tb, and Tc1, Tc2 and Tc3 for reference. Lane1, prestained molecular size markers. The arrows indicate, from top to bottom, phosphorylase b (107 kDa), bovine serum albumin (80 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (26 kDa), and lysozyme (18 kDa).



Physical Properties of Trypsin-activated ChAT

In an effort to elucidate the mechanism by which trypsin activates ChAT, the kinetic properties of the enzyme with respect to substrates, pH/activity profile and salt effects were examined before and after treatment with trypsin. The results of the kinetic analysis of the native and activated ChAT are summarized in Table II. It can be seen that activated ChAT exhibited kinetic properties significantly different from the native enzyme. Trypsin treatment increases the K for both substrates, acetylcholine, and coenzyme A, as well as k. Fig. 4shows the activity of both the native and activated enzyme in buffers containing different concentrations of sodium chloride. The native recombinant enzyme displays an 5-fold enhancement in activity with increasing salt concentration consistent with previous reports of salt effects on this enzyme(29, 30) . However, trypsin-treated ChAT appeared to be maximally activated and did not show any significant salt effect. This suggests that the difference in activity between the trypsin-activated and native forms of the enzyme is dependent on the ionic strength of the assay buffer.


Figure 4: The effect of sodium chloride concentration on enzyme activities of native and proteinase-activated choline acetyltransferase. ChAT activity was measured in the presence of increasing concentrations of sodium chloride at saturating substrate concentrations. Reactions were conducted at 37 °C for 10 min as described under ``Experimental Procedures'': opencircles represent activity of the native choline acetyltransferase, and closedcircles are those for choline acetyltransferase activated with trypsin.



The pH optima of both native and activated enzyme were examined at both low and high ionic strength (Fig. 5). It can be seen that the pH optimum of native ChAT is between pH 6.5 and 7 at the low salt condition, while that of the activated ChAT under the same conditions has been shifted to a value between pH 7.5 and 8. The pH optimum for both the native and trypsin-activated ChAT at high salt are both between pH 7.5 and 8.


Figure 5: Effects of pH on the activity of the native and activated choline acetyltransferase. Enzyme activity of native ChAT in low salt buffer (opencircles) and high salt buffer (opentriangles) and that of ChAT activated with trypsin in low salt buffer (closedcircles) and high salt buffer (closedtriangles). Assays were performed at the indicated pH values at 37 °C for 10 min as described under ``Experimental Procedures.''



Specificity of Activation

To investigate the specificity of activation, different proteinases were tested for their ability to activate ChAT (Table III). The specificity of activation by trypsin was studied by comparing clostripain endoproteinases Arg-C and Lys-C. It is an established fact that trypsin recognizes and cleaves at the carboxyl group of either an arginine or lysine amide bond; endoproteinase Arg-C recognizes and cleaves at the carboxyl group of only the arginine amide bond while endoproteinase Lys-C recognizes and cleaves at the carboxyl group of only the lysine amide bond. Incubation with 100 µg/ml endoproteinase Lys-C activated ChAT, while the same concentration of endoproteinase Arg-C caused inactivation of ChAT. Treatment with equal amounts of both proteinases activated ChAT. This result suggests that treatment with trypsin may cause activation of ChAT and inactivation at the same time, specifically, cleavage at some lysine residue(s) activates ChAT while cleavage at some arginine residue(s) inactivates ChAT. Treatment with chymotrypsin also activates ChAT, suggesting that instead of specific cleavage at certain residue(s) being essential for activation, the cleavages at certain specific regions such as an accessible loop is essential for the activation. Activation of ChAT by trypsin appeared more efficient than by chymotrypsin, although combined incubation with both trypsin and chymotrypsin did not cause further activation. Furthermore, treatment of the trypsin-activated ChAT with chymotrypsin did not affect ChAT activity (data not shown). When the chymotrypsin-treated ChAT was purified away from chymotrypsin and separated on a denaturing SDS-PAGE gel, three bands of 33 kDa Ca, 28 kDa Cb, 21 kDa Cc, were observed (Fig. 6). Compared with trypsin-activated ChAT, there is overall similarity of the nicking/gapping pattern of trypsin-treated ChAT and that of chymotrypsin. There are two peptides (Ta and Ca, Tb, and Cc) of similar sizes generated by either trypsin or chymotrypsin activation as determined by SDS-PAGE gels ( Fig. 3and 6). It is probable that the common region where both trypsin and chymotrypsin cleave could be the event that causes the structural change necessary for ChAT activation. Other proteinases such as papain and protease K did not activate ChAT, on the contrary, they caused irreversible inactivation.


Figure 6: SDS-PAGE analysis of recombinant choline acetyltransferase before and after limited proteolysis with chymotrypsin. Native recombinant rat ChAT (5 µg; lane2) and chymotrypsin-activated ChAT (5 µg; lane3) analyzed on a 10% SDS-PAGE. Proteins were visualized by silver staining. Lane1, prestained molecular size markers as in Fig. 3.



N-terminal Sequence of Trypsin and Chymotrypsin-activated ChAT

Sequence determination of both individual fragments and the mixture of the fragments of the activated ChAT was prepared in order to understand the structural basis for trypsin and chymotrypsin activation. Activated ChAT was separated from either trypsin or chymotrypsin by gel filtration as described under ``Experimental Procedures'' before being denatured and run on a preparative SDS-PAGE gel. The fragments of ChAT were subjected to protein N-terminal sequence analysis, where the sequence of the first 10 amino acids was determined. As shown in Table IV, the sequence of the 31-kDa fragment generated by trypsin treatment is ASSWEELDLP, which corresponds to cleavage at position 15 when compared with the complete amino acid sequence deduced from a cDNA of rat ChAT(22) , while the sequence of the 21-kDa fragment by trypsin treatment is GQLSGQPLXM, which corresponds to cleavage at position 153. The sequence of the 14-kDa fragment by trypsin treatment turned out to yield a mixture of three different fragments. The fragments were consistent with AAMPASEKLQ (481), ADSVSELPAP (363), and ASSWEELDLP(15) . The sequence of the 33-kDa fragment generated by chymotrypsin treatment is EELDLPKLPV, which corresponds to cleavage at position 18. The sequence of the 28-kDa fragment by chymotrypsin treatment, VRADSVSELP, corresponds to cleavage at position 361 and that of the 21-kDa fragment AKGQLSGQPL 151. The trypsin cleavage sites KA (14-15), KG (152-153), RA (362-363), and KA (480-481) are consistent with trypsin specificity. Similarly, the chymotrypsin cleavage sites WE (18-19), WA (150-151), and LV (360-361) are consistent with chymotrypsin specificity. The cleavage sites around amino acids 15-18, 151-153, and 361-363 are in the same regions cut by both trypsin and chymotrypsin, suggesting that those regions are exposed at the surface of ChAT and cleavage of these regions leads to the activation of ChAT. The trypsin cleavage site of AAMPASEKLQ (#481) appears to be unique and therefore probably is not responsible for the activation event. Both trypsin and chymotrypsin-activated ChAT were subjected to N-terminal sequencing as a mixture in order to determine the presence of any small peptide fragments that may not be readily visible and recovered from SDS-PAGE. The results are shown in Table 5, which also contains the different sequences determined from the separated fragments. No new sequences were found, indicating that no small peptides were associated with the activated ChAT. On the other hand, the presence of small gaps cannot be ruled out since only the N-terminal sequence of the fragments were determined. Based on these data, a diagram to illustrate the activated ChAT and its proteolysis sites is shown in Fig. 7.




Figure 7: Schematic representation of the structures of proteolyzed ChAT. The N termini of newly generated ChAT fragments from either chymotrypsin (A) or trypsin (B) cleavage were determined by amino acid sequencing (see Table 4). Cleavage sites are indicated by the designation of the N-terminal amino acid found in each proteolytic fragment, which are labeled as in Table 4with their sizes indicated by dotted lines.






DISCUSSION

Recombinant rat ChAT after trypsin treatment exhibits altered kinetic properties when compared with the native enzyme. The K values for both substrates increase significantly, as does V(max). The observed 56-fold increase in V(max), at a physiological ionic strength, is a minimal estimate since it has been shown with endopeptidase Arg-C that cleavage leading to decreased activity can also occur. Previous studies from this laboratory indicated that the rate-limiting step in the acetylation reaction is primarily the dissociation of the CoA product(30) . Therefore, activation may represent a structural alteration from which the rate of CoA/acetyl-CoA dissociation from the enzyme is increased. Based on its Theorell-Chance mechanism, the K for CoA is k(5)/k(1), and the K for ACh is k(5)/k(3), while kis defined as k(5) (see Fig. S1).


Figure S1: Scheme 1.



The activated enzyme exhibits an approximate 5-10-fold increase in the K value for both of its substrates and a 5-fold increase in the turnover number k(5), consistent with the hypothesis that limited proteolysis causes an increase in the coenzyme A dissociation rate constant, k(5). The mechanism by which salt increases choline acetyltransferase activity has also been proposed to be due to an increase in the coenzyme A dissociation rate(30) .

There are some interesting similarities between salt effects and proteolytic activation of choline acetyltransferase. The pH activity profile of the activated ChAT is very similar to that of the native enzyme in high salt. High salt did not enhance the activity of proteolytically activated ChAT under the same condition in which salt increased the activity of native ChAT. Although high salt does not increase k(5)to the same extent proteolysis does, it seems feasible to suggest that the catalytic residues in proteinase-activated ChAT may experience a similar structural change to those of the native enzyme in high salt. Thus, it seems reasonable to postulate that native choline acetyltransferase is normally in a restrained conformation such that maximal catalytic turnover cannot be achieved and that in the presence of high ionic strength buffer or ``fixed ion pairs'' generated by proteolysis, the conformation of the enzyme undergoes alteration such that the ``optimal'' catalytic capacity is now able to be manifested. The failure to ``super-activate'' trypsin-activated ChAT by chymotrypsin or vise versa appears to agree with the proposed model.

N-terminal sequence analysis of ChAT activated either by trypsin or chymotrypsin indicates that amino acids 14-18, 150-153, 360-363, and 480-481 are localized on the surface accessible to proteinases. Not all cleavages of these bonds are required for activation. For example, digestion of the 480-481 bond appeared unique to trypsin-activated ChAT, suggesting that it is not a trigger for activation. This was proven to be true by mutagenesis in which this trypsin recognition site was changed and trypsin treatment still activated the mutant enzyme. (^3)The cleavage around amino acids 150-153, although present in both trypsin and chymotrypsin-activated ChAT, cannot be responsible for activation. Cleavage of amide bonds around amino acids 150-153 leads to the disappearance of the 31- or 33-kDa bands, which does not result in any further enhancement of ChAT activity. Thus it appears that the cleavage of either amino acids 14-18 and/or 360-363 are required for activation.

The activation of ChAT by proteolysis was also observed with mammalian ChAT purified from brain sources,^3 and therefore is not a result of expression of the recombinant protein. In general, limited proteolysis leading to enzymatic activation can be an important regulatory event in many physiological processes. For example, zymogen activation is a controlled process in terms of temporal and spatial regulation where large amounts of enzyme protein can be initially synthesized and stored in its inactive state and enormous activity can be generated by proteolytic activation in a short period of time or upon release to the location of action. Limited proteolysis has been shown to cause rearrangement of the conformation of the zymogen. This phenomenon is generally seen where regulation at the transcription level is too slow, and reversible mechanisms such as group transfers are not well suited to create a permanent change in the molecular environment. Choline acetyltransferase is synthesized in an active state and it is not known at this time if an in vivo activation mechanism occurs at the site of physiological action by limited proteolysis. However, it is worth noting that in mature sperm, choline acetyltransferase is located in the middle ring of the acrosome followed immediately in cellular space by acrosin, a trypsin-like serine proteinase(31) . At lease three cholinergic markers have been reported to exist in sperm, including the nicotinic receptor acetylcholinesterase and choline acetyltransferase, and these cholinergic components are believed to be involved in sperm maturation and motility(32) . It seems possible that choline acetyltransferase could be activated to increase the rate of biosynthesis of ACh by acrosin. It is of interest to note that Cozzari and Hartman observed both activator(s) and inhibitor(s) of mammalian ChAT(13) . These factors could represent proteinases.

In summary, we have reported a novel proteolytic activation phenomenon for choline acetyltransferase and have addressed the activation mechanism by both kinetic and structural analysis.


FOOTNOTES

*
This work was supported in part by Grant AG05893 from the National Institute on Aging. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Kentucky, College of Medicine, 800 Rose St., Lexington, Kentucky 40536-0084. Tel.: 606-323-6629; Fax: 606-323-1037.

(^1)
The abbreviations used are: ChAT, choline acetyltransferase; PAGE, polyacrylamide gel electrophoresis.

(^2)
D. Wu and L. B. Hersh, manuscript in preparation.

(^3)
D. Wu and L. B. Hersh, unpublished observations.


ACKNOWLEDGEMENTS

We thank the Macromolecule Facility at the University of Kentucky for their service.


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