Prohormone convertase 7 is necessary for the normal processing of cholecystokinin in mouse brain.

Endoproteases in the secretory pathway process pro-cholecystokinin (CCK) into the biologically active forms found in the tissues that express CCK mRNA. Thus far, the endoproteases involved in CCK processing include cathepsin L and the prohormone convertases (PC) 1, 2, and 5. This study finds that PC7 is also critical for normal production of CCK in specific areas of the brain. Loss of PC7 results in decreased levels of CCK in more brain regions than any other endoprotease studied to date. Substantial decreases in brain levels of CCK are found in the prefrontal, frontal, parietal-insular-pyriform, and temporal cortex, caudate-putamen, basal forebrain, thalamus, hippocampus, septum, and medulla of PC7 knock-out (KO) mice. A tissue-specific sexual dimorphism of PC7 activity was also identified. This is the first report that loss of PC7 alters levels of a neuropeptide in the brain. This loss of PC7 and CCK may independently contribute to the decrease in Brain Derived Neurotrophic Factor production and be partially responsible for the learning and memory defects observed in mice that lack PC7.

Characterization of impaired processing of neuropeptides in the brains of endoprotease knockout mice.

With the development of mice in which individual proteolytic enzymes have been inactivated, it has been of great interest to see how loss of these enzymes alters the processing of neuropeptides. In the course of studying changes in the peptide cholecystokinin (CCK) and other neuropeptides in several of these knockout mice, it has become clear that neuropeptide processing is complex and regionally specific. The enzyme responsible for processing in one part of the brain may not be involved in other parts of the brain. It is essential to do a detailed dissection of the brain and analyze peptide levels in many brain regions to fully understand the role of the enzymes. Because loss of these proteases may trigger compensatory mechanisms which involve expression of the neuropeptides being studied or other proteases or accessory proteins, it is also important to examine how loss of an enzyme alters expression of the neuropeptides being studied as well as other proteins thought to be involved in neuropeptide processing. By determining how loss of an enzyme alters the molecular form(s) of the peptide that are made, additional mechanistic information can be obtained. This review will describe established methods to achieve these research goals.

Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties.

Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn(2+)-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX(18)E (Zn(2+)-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G(298)XM(300)E(301)N(302) motif and one mutant of the HEIS(328)HX(18)E motif were expressed in Escherichia coli. All mutations except G(298)P, G(298)S, and S(328)A abolished the aminopeptidase activity. The S(328)A mutant mimics the sequence of bovine Ap-B Zn(2+)-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G(298)S and G(298)P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl(-) anions. Moreover, the G(298)P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G(298) plays in the catalytic mechanism of Ap-B. Our results show that G(298) is essential to Ap-B activity and participates to the substrate specificity of the enzyme.

Cholecystokinin is up-regulated in obese mouse islets and expands beta-cell mass by increasing beta-cell survival.

An absolute or functional deficit in beta-cell mass is a key factor in the pathogenesis of diabetes. We model obesity-driven beta-cell mass expansion by studying the diabetes-resistant C57BL/6-Leptin(ob/ob) mouse. We previously reported that cholecystokinin (Cck) was the most up-regulated gene in obese pancreatic islets. We now show that islet cholecystokinin (CCK) is up-regulated 500-fold by obesity and expressed in both alpha- and beta-cells. We bred a null Cck allele into the C57BL/6-Leptin(ob/ob) background and investigated beta-cell mass and metabolic parameters of Cck-deficient obese mice. Loss of CCK resulted in decreased islet size and reduced beta-cell mass through increased beta-cell death. CCK deficiency and decreased beta-cell mass exacerbated fasting hyperglycemia and reduced hyperinsulinemia. We further investigated whether CCK can directly affect beta-cell death in cell culture and isolated islets. CCK was able to directly reduce cytokine- and endoplasmic reticulum stress-induced cell death. In summary, CCK is up-regulated by islet cells during obesity and functions as a paracrine or autocrine factor to increase beta-cell survival and expand beta-cell mass to compensate for obesity-induced insulin resistance.

Contamination with E1A-positive wild-type adenovirus accounts for species-specific stimulation of islet cell proliferation by CCK: a cautionary note.

We have previously reported that adenovirus-mediated expression of preprocholecystokin (CCK) stimulates human and mouse islet cell proliferation. In follow-up studies, we became concerned that the CCK adenovirus might have been contaminated with a wild-type E1A-containing adenovirus. Here we show conclusively that the proliferative effects reported in the original paper in mouse and human islets were not due to CCK expression but rather to a contaminating E1A-expressing wild-type adenovirus. We also show, however, that CCK expression does have a proliferative effect in rat islets. We hope that our report of the steps taken to detect the wild-type virus contamination, and purification of the contributing viral stocks, will be helpful to other investigators, and that our experience will serve as a cautionary tale for use of adenovirus vectors, especially for studies on cellular replication.

Cathepsin L plays a major role in cholecystokinin production in mouse brain cortex and in pituitary AtT-20 cells: protease gene knockout and inhibitor studies.

Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.

The effects of selective breeding for differential rates of 50-kHz ultrasonic vocalizations on emotional behavior in rats.

Fifty-kHz ultrasonic vocalizations have previously been shown to be positively correlated with reward and appetitive social behavior in rats, and to reflect a positive affective state. In this study, rats selectively bred for high and low rates of 50-kHz vocalizations as juveniles were tested as adults in a battery of behavioral tests for social/emotional behaviors. We found that animals selectively bred for high rates of 50-kHz vocalizations exhibited more crosses into the center area of the open field apparatus, were more likely to show a preference for a dilute sucrose solution (.8%) compared to tap water, and were less aggressive than randomly bred animals. Conversely, animals bred for low rates of 50-kHz calls produced more fecal boli during both open field testing and "tickling" stimulation, and made less contact with conspecifics in a social interaction test compared to randomly bred animals. We also observed that low line rats have elevated brain levels of cholecystokinin (CCK) in the cortex, which is consistent with literature showing that CCK content in the cortex is positively correlated with rates of aversive 22-kHz USVs. Conversely, high line animals had elevated levels of met-enkephalin in several brain regions, which is consistent with the role of endogenous-opioids in the generation 50-kHz USVs and positive affect. These results suggest that animals bred for high rates of 50-kHz may show a stress resilient phenotype, whereas low line rats may show a stress prone phenotype. As such these animals could provide novel insights into the neurobiology of emotion.

Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function.

Type 1 and type 2 diabetes result from a deficit in insulin production and beta-cell mass. Methods to expand beta-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [(3)H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates beta-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating beta-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [(3)H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human beta-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [(3)H]thymidine incorporation. CCK is a beta-cell proliferative factor that is effective in both mouse and human islets. CCK triggers beta-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.

Evidence for defective mesolimbic dopamine exocytosis in obesity-prone rats.

The association between dietary obesity and mesolimbic systems that regulate hedonic aspects of feeding is currently unresolved. In the present study, we examined differences in baseline and stimulated central dopamine levels in obesity-prone (OP) and obesity-resistant (OR) rats. OP rats were hyperphagic and showed a 20% weight gain over OR rats at wk 15 of age, when fed a standard chow diet. This phenotype was associated with a 50% reduction in basal extracellular dopamine, as measured by a microdialysis probe in the nucleus accumbens, a projection site of the mesolimbic dopamine system that has been implicated in food reward. Similar defects were also observed in younger animals (4 wk old). In electrophysiology studies, electrically evoked dopamine release in slice preparations was significantly attenuated in OP rats, not only in the nucleus accumbens but also in additional terminal sites of dopamine neurons such as the accumbens shell, dorsal striatum, and medial prefrontal cortex, suggesting that there may be a widespread dysfunction in mechanisms regulating dopamine release in this obesity model. Moreover, dopamine impairment in OP rats was apparent at birth and associated with changes in expression of several factors regulating dopamine synthesis and release: vesicular monoamine transporter-2, tyrosine hydroxylase, dopamine transporter, and dopamine receptor-2 short-form. Taken together, these results suggest that an attenuated central dopamine system would reduce the hedonic response associated with feeding and induce compensatory hyperphagia, leading to obesity.

Brain regional neuropeptide changes resulting from social defeat.

Past work has demonstrated robust brain changes in cholecystokinin (CCK-8) following social defeat. Here the authors analyzed brain regional, CCK-8, substance P, corticotropin releasing factor (CRF), and neuropeptide Y levels in adult male Long-Evans rats defeated in a resident-intruder social aggression paradigm, as indexed by elevated bites received, freezing, and emission of 20-kHz calls. Brains harvested 6 hr after social defeat were dissected into 12 regions (olfactory bulbs, 3 cortical regions [frontal cortex, cortex above the basal ganglia, cortex above the diencephalon], caudate-putamen, basal forebrain, hypothalamus, hippocampus, thalamus, tectum, tegmentum, and lower brain stem). Neuropeptide radioimmunoassays demonstrated the following statistically significant regional changes in defeated rats as compared with nondefeated rats: CCK-8 was reduced in frontal cortex and cortex overlying diencephalon, the olfactory bulbs, caudate-putamen, hippocampus, tectum, and lower brainstem. Neuropeptide Y was elevated in the caudate-putamen. Substance P was elevated in the cortex over the basal ganglia and decreased in basal forebrain. CRF was diminished in the hippocampus. The results highlight more robust CCK modulation by social defeat as compared with 3 other neuropeptide systems involved in brain emotional regulation.

Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling.

BACKGROUND: Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. RESULTS: The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. CONCLUSION: Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.

Regional brain cholecystokinin changes as a function of rough-and-tumble play behavior in adolescent rats.

Brain cholecystokinin (CCK) levels have been shown to be elevated in animals defeated during adult social aggression. The present experiment evaluated whether similar effects are evident in prolonged bouts of juvenile social-play fighting, which tend to switch from largely positive to some negative affect after approximately 15 min into a half-hour play session, as indexed by a gradual shift from positively valenced 50 kHz ultrasonic vocalizations (USVs) to negatively valenced 20 kHz USVs. Given the role of CCK in both positive and negative emotional events, we examined levels of CCK-8 in tissue homogenates from 14 brain areas in animals 6h after a 30 min play bout compared to no-play control animals tested similarly in isolation for 30 min. As with patterns observed following adult defeat, significantly higher CCK levels were evident after play in the posterior neo-cortex compared to no-play control animals (+26%). Levels of CCK were also elevated in the midbrain (+35%). However, unlike in adult aggression, CCK levels were reduced in the hypothalamus (-40%) and basal forebrain (-24%) as compared to no-play animals. Posterior cortex CCK levels were positively correlated to the duration that each animal was pinned (r = +.50) which suggests that elevated CCK in the posterior cortex may be related to the negative aspects of play. Hypothalamic CCK levels were negatively related to dorsal contacts and pins (r's = -.57), and suggest that the lower CCK levels may reflect the more positive valenced aspects of play. The data indicate that CCK utilization in the brain is dynamically responsive to rough-and-tumble play.

Inhibition of PC5 expression decreases CCK secretion and increases PC2 expression.

Cholecystokinin (CCK) is produced from pro CCK by a series of enzymatic cleavages. One of the enzymes thought to be important for pro CCK cleavage is prohormone convertase 5 (PC5). STC-1 cells, a mouse intestinal tumor cell line that expresses CCK, PC1, PC2, and PC5 were stably transfected with hairpin loop plasmids encoding siRNA targeting PC5 and clones were selected. CCK secretion was reduced significantly. PC5 mRNA and protein expression as measured by quantitative PCR and Western blot analysis was reduced about 50%. CCK and PC1 mRNA expression were not changed. These cells showed a three-fold increase in PC2 mRNA and protein expression. This increase may represent a compensatory mechanism triggered by the loss of PC5. The decrease in CCK in the media was due largely to loss of CCK 22. These results provide the first direct evidence that PC5 is involved in CCK processing.

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations.

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.

Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide.

Purified recombinant prohormone convertase 1 and 2 (PC1 and PC2) cleave a peptide containing cholecystokinin (CCK) 8 Gly Arg Arg and the carboxyl-terminal peptide liberating CCK 8 Gly Arg Arg. PC1 and PC2 also cleave purified pro CCK, liberating the amino terminal pro-peptide while no carboxyl-terminal cleavage was detected. Under the conditions of the in vitro cleavage assay, it appears that the carboxyl-terminal cleavage site of pro CCK is not accessible to the enzymes while this site is readily cleaved in a synthetic peptide. Additional cellular proteins that unfold the prohormone may be required to expose the carboxyl-terminal site for cleavage.

Regional brain cholecystokinin changes as a function of friendly and aggressive social interactions in rats.

Cholecystokinin (CCK) is the most abundant neuropeptide in the mammalian brain, and has been implicated in the regulation of a diversity of emotions and motivations including negative affect and stress responses. In this experiment, we assayed levels of CCK (CCK4/5 and CCK8) from tissue homogenates in intruder animals 6 h after resident-intruder inter-male aggression. Intruder animals that demonstrated submissive behavior (freezing and 22-kHz ultrasonic vocalizations) had higher levels of CCK in the tegmentum and posterior cortex as compared to non-submissive (i.e., "Friendly") intruder animals. Ultrasonic vocalizations (22-kHz) were positively correlated with CCK levels in the tegmentum, posterior cortex and pituitary. These data suggest that CCK may play a role in the generation of negative affective states indexed by 22-kHz ultrasonic calls in certain regions of the brain.

Cleavage-site mutagenesis alters post-translation processing of Pro-CCK in AtT-20 cells.

Cholecystokinin (CCK) is expressed in the central and peripheral nervous systems and functions as a neurotransmitter and neuroendocrine hormone. The in vivo forms of CCK include CCK-83, -58, -39, -33, -22, -12, and -8. Tissues in the periphery produce the larger forms of CCK, such as CCK-58, whereas the brain primarily produces CCK-8. The different biologically active forms of CCK observed in vivo may result from cell-specific differences in endoproteolytic cleavage during post-translational processing. Evidence suggests that cleavages of pro-CCK occur in a specific sequential order. To further delineate the progression of cleavages during pro-CCK maturation, mutagenesis was used to disrupt putative mono- and dibasic cleavage sites. AtT-20 cells transfected with wild-type rat prepro-CCK secret CCK-22 and -8. Mutagenesis of the cleavage sites of pro-CCK had profound effects on the products that were produced. Substitution of basic cleavage sites with nonbasic amino acids inhibits cleavage and leads to the secretion of pathway intermediates such as CCK-83, -33, and -12. These results suggest that CCK-58 is cleaved to both CCK-33 and -22. Furthermore, CCK-8 and -12 are likely derived from cleavage of CCK-33 but not CCK-22. Alanine substitution at the same site completely blocked production of amidated products, whereas serine substitution did not. The cleavages observed at nonbasic residues in this study may represent the activity of enzymes other than PC1 and carboxypeptidase E, such as the enzyme SKI-1. A model for the progression of pro-CCK processing in AtT-20 cells is proposed. The findings in this study further supports the hypothesis that pro-CCK undergoes parallel pathways of proteolytic cleavages.

Expression and purification of rat recombinant aminopeptidase B secreted from baculovirus-infected insect cells.

Aminopeptidase B (Ap-B) is a ubiquitous enzyme and its physiological function still remains an open question. This Zn2+ -exopeptidase catalyzes the amino-terminal cleavage of basic residues of peptide or protein substrates, indicating a role in precursor processing. In addition, the enzyme exhibits a residual capacity to hydrolyze leukotriene A4 (LTA4) into the pro-inflammatory lipid mediator leukotriene B4 (LTB4) in vitro. This potential bi-functional nature of Ap-B is supported by a close structural relationship with LTA4 hydrolase, which hydrolyzes LTA4 into LTB4, in vivo, and exhibits an aminopeptidase activity, in vitro. Structural studies are necessary for the detailed understanding of the bi-functional enzymatic mechanism of Ap-B. In this study, we report cDNA cloning, baculovirus expression, and purification of the rat Ap-B (rAp-B). The Ap-B cDNA was constructed from extracted rat testes total RNA and introduced into the pBAC1 baculovirus transfer vector to generate recombinant baculoviruses. rAp-B expression, with or without COOH-hexahistidine tag, was tested in two different insect cell hosts (Sf9 and H5). The enzyme is secreted into the insect cell culture medium, which allowed a rapid purification of the protein. The His-tagged rAp-B was purified using metal affinity resin while the native recombinant rAp-B was partially purified using a single step DEAE Trisacryl ion exchange column. Although the recombinant rAp-B exhibits biochemical properties equivalent to those of the rat testes purified protein, the presence of the histidine-tag seems to partially inhibit the exopeptidase activity. However, this report shows that baculovirus-infected cells are a useful system to produce rat Ap-B for use in studying enzymatic mechanisms in vitro and 3D structure.