Levels of Small Molecules and Enzymes in the Mother Cell Compartment and the Forespore of Sporulating Bacillus megaterium

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.     

Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.     

Release and recovery of forespores from Bacillus cereus

A method is described which makes possible the release of immature forespores from sporulating cells at specific stages of development, from the completion of stage III through to mature spore formation. With the aid of zonal density gradient centrifugation, the method makes possible the recovery of quantities of forespores ample for biochemical and physical studies. With the capability to examine forespores and some mother cell components independently, we have established that several enzymes associated with the sporulation process are localized in the newly developed forespores. Studies showed that aspartate aminotransferase and alanine aminotransferase are associated with the forespores, whereas l-alanine dehydrogenase is found only in the mother cell cytoplasm.     

The red-ox status of a penicillin-binding protein is an on/off switch for spore peptidoglycan synthesis in Bacillus subtilis

Thiol-disulphide oxidoreductases catalyse the formation or breakage of disulphide bonds to control the red-ox status of a variety of proteins. Their activity is compartmentalized, as exemplified by the distinct roles these enzymes play in the cytoplasm and periplasm of Gram-negative bacteria. In this issue of Molecular Microbiology , an article from Lars Hederstedt and collaborators at Lund University sheds light on another member of this superfamily of proteins, the thioredoxin-like protein StoA from Bacillus subtilis . Interestingly, StoA function is required in yet another subcellular compartment: the intermembrane space that separates forespores from mother cells in endospore-forming bacteria. Specifically, this study demonstrates that the high-molecular-weight penicillin-binding protein SpoVD, which contains two exposed cysteine residues and whose extracellular domain is located in the intermembrane space, is a substrate of StoA. As formation of a disulphide bond most likely inactivates SpoVD activity, the converse breakage of that bond in a process catalysed by StoA appears to be the trigger that initiates peptidoglycan synthesis in sporulating cells.     

Respiratory systems of the Bacillus cereus mother cell and forespore.

The respiratory systems of the mother cells and forespores of Bacillus cereus were compared throughout the maturation stages (III to VI) of sporulation. The results indicated that both cell compartments contain the same assortment of oxidoreductases and cytochromes. However membrane fractions from young forespores were clearly distinct from those of the mother cell, i.e., lower content of cytochrome aa3, lower cytochrome c oxidase activity, higher concentration of cytochrome o, and a lower sensitivity of the respiration to the inhibiting effect of cyanide. This suggests that the cyanide-resistant pathway contributes more importantly to forespore respiratory activity than to activity in the mother cell compartment. During the maturation stages, the forespore NADH oxidase activity declined faster than in the mother cells. Other activities studied decreased steadily in both cell compartments. These findings together with the analysis of the kinetics of NADH-dependent reduction of cytochromes in the mature spore membranes indicated an impairment of electron flow between NADH dehydrogenase and cytochrome b. This impairment could be overcome by the addition of menadione.     

Localization of 3-phosphoglyceric acid synthesis in the mother cell compartments and forespores ofBacillus megaterium and the effects of manganous ions on its metabolism

Rapidly metabolizable compounds such as glucose or glycerol were not utilized byBacillus megaterium in the absence of manganese when grown in the supplemented nutrient broth medium. Under these conditions, growth ceased at low cell titre, 3-phosphoglyceric acid accumulated inside the cells and normal sporulation process was arrested. Addition of manganese to the medium caused disappearance of 3-phosphoglyceric acid, growth resumed and normal sporulation was observed. Synthesis of 3-phosphoglyceric acid occurred only in the mother cell compartments and it was transported for accumulation inside the forespores ofBacillus megaterium when grown in supplemented nutrient broth medium. Incubation of forespores in the presence of glucose or glycerol had no effect on 3-phosphoglyceric acid synthesis/accumulation, but it was completely utilized when forespores were incubated with manganese plus ionophore (X 537A). No other metal(s) could substitute for manganese suggesting that manganese plays crucial role in 3-phosphoglyceric acid metabolism     

Subcellular localization and levels of aminopeptidases and dipeptidase in Saccharomyces cerevisiae.

Three aminopeptidases (L-aminoacyl L-peptide hydrolases, EC 3.4.11) and a single dipeptidase (L-aminoacyl L-amino acid hydrolase, EC 3.4.13) are present in homogenates of Saccharomyces cerevisiae. Bassed on differences in substrate specificity and the sensitivity to Zn2+ activation, methods were developed that allow the selective assay of these enzymes in crude cell extracts. Experiments with isolated vacuoles showed that aminopeptidase I is the only yeast peptidase located in the vacuolar compartment. Aminopeptidase II (the other major aminopeptidase of yeast) seems to be an external enzyme, located mainly outside the plasmalemma. The synthesis of aminopeptidase I is repressed in media containing more than 1% glucose. In the presence of ammonia as the sole nitrogen source its activity is enhanced 3--10-fold when compared to that in cells grown on peptone. In contrast, the levels of aminopeptidase II and dipeptidase are less markedly dependent on growth medium composition. It is concluded that aminopeptidase II facilitates amino acid uptake by degrading peptides extracellularly, whereas aminopeptidase I is involved in intracellular protein degradation.     

Characterization and deposition of the proteins in the outermost layer of Bacillus megaterium spore

It was proved that three spore coat proteins of 48, 36, and 22 kDa (P48, P36, and P22) were the components of the outermost layer (OL) of Bacillus megaterium ATCC 12872 spore by analysis of the isolated OL. And it was indicated that these proteins were deposited not by disulfide bond, but by ionic and/or hydrophobic bonds on the spore. Among them, P36 and P22 were expected to be located on the very surface of the spore by immunological analysis. In the OL deficient mutant of B. megaterium ATCC 12872, MAE05, whose spore was lacking in these OL proteins and galactosamine-6-phosphate polymer, both P36 and P22 were present in the mother cell cytoplasm and deposited on the forespores, but they disappeared with the lysis of mother cells. An OL protein-releasing factor having proteolytic activity was detected in the culture supernatant at the late sporulating stage of both the wild-type and the mutant strains. But the factor could not act on the proteins of the mature spores and the forespores at t10 (tn indicates n hr after the end of exponential growth) of the wild-type strain. Moreover, P36 and P22 were found in the spores of a revertant of MAE05 which could form galactosamine-6-phosphate polymer, suggesting that this sugar polymer played the role in protecting the OL proteins against the protease-like substance after the deposition.     

The influence of the developing bacterial spore on the mother cell

Mutants unable to develop a completely engulfed forespore do not lose their viability, i.e., their ability to resume cell division, for at least 10 hr after the end of exponential growth. In contrast, mutants, which are blocked at later stages in development and which are able to produce completely engulfed forespores, lose their ability to divide. The time course of this decrease in viability coincides with the time course for the appearance of completely enclosed forespores. Experiments with the sporulating standard strains of Bacillus subtilis and B. megaterium suggest that the mother cells also lose their viability at about the time of forespore enclosure. These results indicate that the forespore, as soon as it is completely engulfed and thus committed to continue differentiation, somehow prevents the mother cell (sporangium) from resumption of growth.     

Analysis of glycan polymers produced by peptidoglycan glycosyltransferases

Bacterial cells are surrounded by a cross-linked polymer called peptidoglycan, the integrity of which is necessary for cell survival. The carbohydrate chains that form the backbone of peptidoglycan are made by peptidoglycan glycosyltransferases (PGTs), highly conserved membrane-bound enzymes that are thought to be excellent targets for the development of new antibacterials. Although structural information on these enzymes recently became available, their mechanism is not well understood because of a dearth of methods to monitor PGT activity. Here we describe a direct, sensitive, and quantitative SDS-PAGE method to analyze PGT reactions. We apply this method to characterize the substrate specificity and product length profile for two different PGT domains, PBP1A from Aquifex aeolicus and PBP1A from Escherichia coli. We show that both disaccharide and tetrasaccharide diphospholipids (Lipid II and Lipid IV) serve as substrates for these PGTs, but the product distributions differ significantly depending on which substrate is used as the starting material. Reactions using the disaccharide substrate are more processive and yield much longer glycan products than reactions using the tetrasaccharide substrate. We also show that the SDS-PAGE method can be applied to provide information on the roles of invariant residues in catalysis. A comprehensive mutational analysis shows that the biggest contributor to turnover of 14 mutated residues is an invariant glutamate located in the center of the active site cleft. The assay and results described provide new information about the process by which PGTs assemble bacterial cell walls.     

Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli.

In an attempt to bring some insight into how peptidoglycan synthesis is controlled in Escherichia coli, simple parameters, such as cell peptidoglycan content, the pool levels of its seven uridine nucleotide precursors, and the specific activities of five enzymes involved in their formation, were investigated under different growth conditions. When exponential-phase cells with generation times ranging from 25 to 190 min were examined, the peptidoglycan content apparently varied as the cell surface area changed, and no important variations in the pool levels of the nucleotide precursors or in the specific activities of the five enzymes considered were observed. The peptidoglycan of exponential-phase cells accounted for 0.7 to 0.8% of the dry cell weight, whereas that of stationary-phase cells accounted for 1.4 to 1.9%. Depending on the growth conditions, the number of peptidoglycan disaccharide peptide units per cell varied from 2.4 X 10(6) to 5.6 X 10(6). The levels of the nucleotide precursor pools as well as the specific activities of the D-glutamic acid- and D-alanyl-D-alanine-adding enzymes varied little with the growth phase. The specific activities of UDP-N-acetylglucosamine transferase, UDP-N-acetylglucosamine-enolpyruvate reductase, and the diaminopimelic acid-adding enzymes decreased by 20 to 50% at most in the late stationary phase. The results are discussed in terms of the possible importance for cell survival of the maintenance of a high capacity for peptidoglycan synthesis, whatever its rate under various growth conditions, and of a balance between the synthesis and breakdown of peptidoglycan during active growth.     

Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.     

Characterization of a polysaccharide deacetylase gene homologue (pdaB) on sporulation of Bacillus subtilis

The predicted amino acid sequence of Bacillus subtilis ybaN (renamed pdaB) exhibits high similarity to those of several polysaccharide deacetylases. Northern hybridization analysis with sporulation sigma mutants indicated that the pdaB gene is transcribed by EsigmaE RNA polymerase and negatively regulated by SpoIIID. The pdaB mutant was deficient in spore formation. Phase- and electron microscopic observation showed morphological changes of spores in late sporulation periods. The pdaB spores that had lost their viability were empty. Moreover, GFP driven by the promoter of the sspE gene was localized in the forespore compartment for the wild type, but was localized in both the mother cell and forespore compartments for phase-gray/dark forespores of the pdaB mutant. This indicates that GFP expressed in the forespores of the mutant leaks into the mother cells. Therefore, PdaB is necessary to maintain spores after the late stage of sporulation.     

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.     

Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.     

The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.     

Identification of substrates of the Listeria monocytogenes sortases A and B by a non-gel proteomic analysis

Sortases are enzymes that anchor surface proteins to the cell wall of Gram-positive bacteria by cleaving a sorting motif located in the C-terminus of the protein substrate. The best-characterized motif is LPXTG, which is cleaved between the T and G residues. In this study, a non-gel proteomic approach was used to identify surface proteins recognized by the two sortases of Listeria monocytogenes, SrtA and SrtB. Material containing peptidoglycan and strongly associated proteins was purified from sortase-defective mutants, digested with trypsin, and the resulting peptide mixture analysed by two-dimensional nano-liquid chromatography coupled to ion-trap mass spectrometry. Unlike enzymes involved in peptidoglycan metabolism, other surface proteins displayed uneven distribution in the mutants. A total of 13 LPXTG-containing proteins were identified exclusively in strains having a functional SrtA. In contrast, two surface proteins, Lmo2185 and Lmo2186, were identified only when SrtB was active. The analysis of the peptides identified in these proteins suggests that SrtB of L. monocytogenes may recognize two different sorting motifs, NXZTN and NPKXZ. Taken together, these data demonstrate that non-gel proteomics is a powerful technique to rapidly identify sortase substrates and to gain insights on potential sorting motifs.     

Inhibition by bestatin of a mouse ascites tumor dipeptidase. Reversal by certain substrates

Bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl]-L-leucine, a known inhibitor of aminopeptidases, is shown to be a potent linear competitive inhibitor (KI,2.7 nM) of a dipeptidase purified from Ehrlich-Lettré hyperdiploid mouse ascites tumor cells. This inhibition can be classified as "slow binding" but not as "tight binding." Substrate protects the enzyme from bestatin inhibition when enzyme and inhibitor are in approximately equimolar concentrations. Addition of substrate (6 mM) partially (by about 20%) reverses dipeptidase inhibition by bestatin, but the time required for maximum recovery depends on the nature of the substrate. Substrates with lower Km (0.28-1.4 mM) values that exhibit substantial substrate inhibition require longer times (23-65 min) than those with higher Km values that show little substrate inhibition. Substrates with Km values higher than 1.5 mM do not reverse inhibition. The inhibition of the tumor dipeptidase by bestatin has been compared with inhibition by a variety of inhibitors of other Zn-metallo-proteolytic enzymes. These inhibitors were far less potent (KI, 0.063-10 mM), indicating a difference between the tumor dipeptidase and other enzymes of that class. Our results are discussed in terms of a postulated model of the bestatin molecule in the active site of the tumor dipeptidase, an enzyme which has not been studied by x-ray crystallographic means. The phenyl group of bestatin is placed in a hydrophobic pocket that is external but adjacent to the active site of the tumor dipeptidase. The shape of this pocket, as it appears from our results plus modeling, is such that only certain R groups of substrate can fit. The existence of such a pocket might explain the differential effect of substrates in the reversal of bestatin inhibition of the dipeptidase and also might explain substrate inhibition by misalignment of R groups into this pocket.     

Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.