Protein Digestion and Absorption

Vol. 61, No. 4, Part 1

GASTROENTEROLOGY

Copyright © 1971 by The Williams & Wilkins Co.

Printed in U.S. A.

PROGRESS IN GASTROENTEROLOGY PROTEIN DIGESTION AND ABSORPTION GARY

M.

GRAY, AND HERBERT

L.

CooPER

Division of Gastroenterology, Stanford University School of Medicine, Stanford, California, and Division of Gastroenterology, University Hospital, Boston, Massachusetts

The processes whereby protein is digested and absorbed are not as well understood as are those for other nutrients but, nevertheless, a great deal of information has come forth in the last few years. As has been true in the study of assimilation of carbohydrates and fat, the focus of interest has been on the function of the intestinal cell and its role in transporting the amino acid products of digestion. Despite this, it is important to consider the over-all processes of digestion and absorption and this review considers both the pancreatic and small intestinal phases of protein digestion and absorption.

Pancreatic Enzyme Secretion More than 20 years ago several of the proteolytic enzymes had been isolated in essentially pure form from bovine and porcine pancreas, ,_ 3 and it was determined that the proteases were secreted into the duodenum as inactive precursors. Since then, the actual structure of many of these enzymes in both the proenzyme and active state has been elucidated. 4 - 6 There are two general classes of pancreatic proteases. The endopeptidases trypsin, chymotrypsin, and elastase attack the interior peptide (CO-NH) bonds between adjacent amino acids and differ Received October 17, 1969. Address requests for reprints to: Dr. Gary M. Gray, Division of Gastroenterology, Stanford University School of Medicine, Stanford, California 94305. This work was supported by United States Public Health Service Grant AM11270. Dr. Gray is a recipient of Research Career Development Award 1-K4-AM-47,443.

functionally from each other only in their specificity for particular amino acids. The principal exopeptidases are the carboxypeptidases which act on terminal peptide bonds at the carboxyl (COOH) end of protein chains. Both the endo- and exopeptidases are synthesized in the pancreatic acinar cells and then secreted as inactive precursors via the pancreatic ducts into the upper duodenal lumen where activation occurs by a specific scission of the protein molecule. Keller's comprehensive review 7 of the activation, structure, and specificity of pancreatic proteases may be useful to the reader. wishing more details than are provided below.

Intraduodenal Activation of Pancreatic Pro teases Endopeptidases. Although the majority of data available on the pancreatic proteases has come from study of the bovine and porcine enzymes, differences from species to species appear to be small and man can be expected to possess very similar structures for the enzymes. As outlined in figure 1, the proenzymes or zymogens are secreted into the duodenal lumen where initial activation of trypsinogen is catalyzed by enterokinase, an enzyme located on the surface of the duodenal mucosa. 8 The exact structure of this activating protein is unknown, but it splits a fragment from the amino terminal end of the trypsinogen molecule by hydrolyzing the peptide bond between lysine-6 and isoleucine-7 thereby releasing a hexapeptide and the active trypsin (fig. 2). After this initial acti535

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Vol1 - - L y s 6 vation, the active trypsin acts autocatalyticVoi1-Lys 6fi1o 7) ally in the same way as enterokinase to activate the bulk of the trypsinogen. As C ' - - - - - Asn 2 29 ' - - - - - Asn 229 outlined in figure 1, trypsin then activates TRYPSINOGEN all of the other precursor peptidases of the pancreas including chymotrypsinogen, proelastase, and the procarboxypeptidases. Cys 1 - - ArQ t5 flle 16 Cys 1- - A r g 15 Chymotrypsinogen, being very similar J~ 22-~~· ·· in structure to trypsinogen, 7 is activated by relatively small amounts of trypsin (one part 11 CHYMOTRYPSIN CHYMOTRYPSINOGEN in 70) 9 which splits the CO- NH bond beFIG. 2. Diagram of the mechanism of activation tween arginine-15 and isoleucine-16 (fig. of bovine pancreatic proenzymes. Abbreviations for 10 2). Although a structural rearrangement amino acids units are: Val, valine; Lys, lysine; Ile, isothen occurs, no peptide fragments are leucine; Asn, asparagine; Cys, Cysteine. The numberactually released because there is a disul- ing refers to the position on the protein chain beginning fide bond between cysteine-1 and cysteine- at the N-terminal amino acid. 122 (fig. 2). This form of the enzyme is called 1r-chymotrypsin. Secondary cleav- activation of the exopeptidases is still relaages at other parts of the molecule also tively rudimentary. The exact alteration occur by autocatalytic means producing in their structure that is necessary for actithe other species (usually termed a and vation is not known. Under physiological {3) of chymotrypsin with relatively less conditions of temperature and trypsin conactivity which may not play a physiolog- centration, carboxypeptidase A is formed ical role in protein digestion. when its parent zymogen procarboxypepProelastase, the inactive zymogen, be- tidase A is extensively degraded by the comes activated by trypsin to elastase, an action of trypsin. The active carboxypepenzyme with broad specificity against in- tidase A is only about one-third the size of terior peptide bonds of proteins and the its precursor. I I only protease that can attack elastin (fig. Procarboxypeptidase B is also activated 1). Although the exact mechanism of acti- by trypsin but, unlike the other active exovation is unknown, the amino acid sequence peptidase, carboxypeptidase B is either of the active enzyme itself has recently the same molecular size (during early actibeen elucidated. 6 Its structure is remark- vation) or slightly smaller (second phase of ably similar to trypsin and chymotrypsin. activation) than its zymogen precursor. I 2 The differences in specificity of these Substrate Specificity of Pancreatic three types of endopeptidases is outlined Pro teases below and appears to occur because of a few selective amino acid differences at Because of the restricted site of action the substrate sites accompanied by exten- of many of the proteases, protein digestion sive but relatively unimportant substitu- occurs only by virtue of a sequential action tions at other portions of the enzyme. 6 of specific endopeptidase and exopeptidase Exopeptidases. Information about the (fig. 3). Hence trypsin hydrolyzes protein to peptides only at locations of the basic PROENZYME ENZYME amino acids thereby yielding peptides with only arginine or lysine on the carboxyl terTRYPSINOGEN TRYPSIN minal end of the molecule. 13 CarboxyCHYMOTRYPSINOGEN CHYMOTRYPSIN peptidase B can then hydrolyze the single l arginine or lysine from the C-terminal poPROELASTASE ELASTASE sition since it has high specificity for these I CARBOXYPEPTIDASE PROCARBOXYPEPTIOASE basic amino acids. 14 On the other hand, chymotrypsin acts FIG. 1. Activation of the pancreatic proteases in the duodenal lumen. Modified from Keller. 7 interiorly at aromatic amino acid sites to ENTEF«>I
L. ,22

c

-=

c

cL.. 7

[NT[ROKINASE

I

TRYPSIN

TRYPSIN

TRYPSIN

--

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PROGRESS IN GASTROENTEROLOGY

produce peptides with a C-terminal phenylalanine, tyrosine, or tryptophan; in addition some chymotrypsins also have specificity for leucine, glutamine, and methionine. 15 Elastase has the broadest specificity of any endopeptidase acting particularly on the aliphatic (nonpolar) amino acids such as valine, leucine, serine, and alanine to produce peptides with these amino acids at the carboxy-terminus. 6· 16 These aromatic and aliphatic C-terminal peptides produced by chymotrypsin and elastase are ideal substrates for carboxypeptidase A, 7 which hydrolyzes the neutral amino acid from the C-terminus. Figure 3 outlines the concerted action of pancreatic endopeptidases and exopeptidases.

Intestinal Oligopeptidases: Brush Border and Intracellular Like the disaccharidases, enzymes hydrolyzing small peptides are present in only trace amounts in pancreatic or intestinal secretions' 7 and appear to be confined to the intestinal surface cell. 18 · 19 There has been much controversy concerning the digestion and absorption of the smaller molecular products of pancreatic enzyme digestion. A principal reason for this is the finding of oligopeptidases both in the interior of the small intestinal columnar cell' 8 as well as within its luminal brush border, 20 making it difficult to determine whether the small peptides are split on the outer surface of the intestinal cell or transported ENDOPEPTIDASES

EXOPEPTIDASE$

BASIC

AMINO ACIDS TRYPSIN

PROTEIN

CHYMOTRYPSIN

At g!IC,oC · TEII- ~

PEPTIOES

CAfiBOXYPEPTIOASE

8

AIIOioiATIC C·f(IUIIHAl

SMALL PEPTIOES

PEPTIOES

CARBOXYPEPTIDASE A

ELASTASE

NEUTRAL PEPTIOES

AMINO ACIDS

FIG. 3. lntraduodenal sequential action of pancreatic endopeptidases and exopeptidases on dietary protein. The final products on the right are the substrates which must be handled by the intestinal cell. Arg, arginine; Cys, cysteine.

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across the luminal cell boundary to be hydrolyzed by intracellular enzymes. However, recent studies of the brush border and intracellular enzymes indicate that the brush border enzymes are distinctly different from the intracellular peptidases, 21 · 22 and the soluble peptidases appear to be no different than enzymes that occur within cell sap of many other organs. 22, 23 Intracellular peptidases. Nevertheless, the intracellular (soluble) peptidases probably have the capacity to hydrolyze small peptides that enter the cell in intact form. The bulk of the work on intestinal peptidases in animals and man has been on these soluble enzymes that are located in the cell cytoplasm and account for about 80% of the total mucosal activity of most peptide hydrolases that have been studied. 18· 19· 24 -27 There appear to be four to eight enzymes separable by starch gel electrophoresis that can hydrolyze various dipeptides and tripeptides. 28 Whereas glycine- and leucinecontaining peptides have been studied most extensively, other peptides containing an aliphatic or aromatic neutral amino acid are readily hydrolyzed by these soluble enzymes. (Naturally occurring amino acids in dietary protein are structurally of the L-form. We have chosen to omit the designation, but whenever an amino acid is mentioned in peptide or in free acid, it indicates the L isomer rather than the D isomer.) In addition, there are soluble peptidases that hydrolyze glycyl-glycine 19 · 25 and dipeptides containing proline29 as well as an enzyme that is highly specific for N-terminal arginine or lysine. 24 Brush border peptidases. Although the brush border peptidases have not yet been studied extensively, 10 to 20 ~c of intestinal dipeptidase activity is known to be present in the microvillar membrane. 20 · 30 These enzymes appear to be distinctly different proteins than those found in soluble form. 21 · 22 Recently three brush border peptidases have been separated and characterized in the rat with specific activities averaging 10 times higher than those found for the intracellular enzymes (F. Wojnarowska and G. M . Gray, unpublished observa-

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tions). Two of these show specificity for dipeptides, tripeptides, and tetrapeptides composed of neutral amino acids on the amino-terminal end with maximal activity exerted against the tripeptides. These are probably best termed oligopeptidases. The other major brush border peptidase seems to be a true dipeptidase that has minimal specificity for larger oligopeptides. Like the oligopeptidases, it acts preferentially against dipeptides composed of neutral amino acids, particularly glycyl-leucine and leucyl-glycine. Activity against prolinecontaining peptides is absent for all three of these enzymes. Hence, different peptidases appear to be strategically located for digestion both in the brush border and within the intracellular fluid. Like the disaccharidases, the brush border peptidases are ideally located at the intestinal mucosa-lumen interface and have specificity appropriate for small peptides composed of neutral amino acids. However, the brush border enzymes seem incapable of rapidly hydrolyzing proline and hydroxyproline peptides or arginyl and lysyl peptides whereas the intracellular peptidases are more active against these substrates 24 • 29 making it likely that the cell surface and the interior cytoplasm play a complementary role m digestion of the oligopeptides.

Digestion of Dietary Protein: In Vivo Studies Whether oligopeptides and dipeptides enter the intestinal cell in intact form or rather are initially hydrolyzed to amino acids can only be interpreted in the light of physiological experiments. Previous studies in animals and man 3 1 had suggested that an appreciable fraction of the protein in intestinal contents after a meal was made up of endogenous proteins. More recent experiments in man 32 • 33 provide convincing evidence that 65 to 90S;, of the postprandial intraintestinal protein is exogenous in its origin making it much more feasible to interpret in vivo absorption studies by determining the degree of disappearance of protein and the size of residual peptides. As estimated from a thorough examina-

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tion of the composition of intestinal contents after oral intake of protein, Nixon and Mawer 32 • 33 demonstrated that man hydrolyzes milk or gelatin within 15 min to 30 to 50% free amino acids and small peptides with the remainder of the products being in the form of large peptides and protein. The same fractional distribution pattern continues during the digestion process for at least 2 hr as the meal moves well down into the jejunum. The vast majority of the milk or gelatin meal is hydrolyzed and absorbed by the time the meal reaches the distal jejunum. Interestingly enough, only certain amino acids are readily released in the free form. Appreciable amounts of the basic amino acids (arginine, lysine) and neutral amino acids (valine, phenylalanine, tyrosine, methionine, leucine) are released to be transported by the intestinal cell. In contrast, glycine, the imino acids (proline, hydroxyproline), the hydroxyl-substituted amino acids (serine and threonine), and the dicarboxylic amino acids (aspartic and glutamic) continue to remain about 90Sr peptide linked in the intestinal lumen until they disappear and hence probably enter the intestinal cell as constituents of small peptides; the alternative explanation of hydrolysis of such oligopeptides by brush border surface enzymes followed by complete capture by the amino acid transport mechanisms seems highly unlikely since surface digestion of nutrients is known to be associated with considerable diffusion of released products such as hexoses 3 4 or amino acids 35 • 36 back into the intestinal fluids . Other studies in both animals 36 • 37 and man 38 have compared absorption from solutions of peptides to that of equivalent amino acid mixtures, particularly by use of glycine peptides (glycyl-glycine and glycylgylcyl-glycine) and glycine. Evidence clearly indicates that glycine is absorbed more rapidly from the dipeptide and tripeptide, suggesting that the small peptides probably enter the cell at rates comparable to the amino acid, presumably at the same entry sites. 37 Thus three molecules of glycine can move into the cell more efficiently at a specific entry site as constituents of a single tripeptide molecule rather

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than as three molecules of the amino acid. Supporting the absorption of intact glycine peptides is the finding that intestinal peptidase activity against them in the brush border is only 1% of the sucrase activity. 20 In vitro studies of isolated rat intestine also show absorption of intact gylcine peptide followed by intracellular hydrolysis of 90% and exit of 10% from the cell as intact dipeptide. 39 Recently, this has been corroborated in vivo in the rat 40 by demonstration of glycyl-glycine in portal venous blood at concentrations 4 to 8% of that for glycine during absorption of ~lycine peptides. Despite these interesting studies with oligopeptides of glycine, dietary protein rarely contains a series of glycine residues and the physiological importance of such oligopeptide absorption is uncertain. Analogous to the findings for glycine peptides, about 10% of a gelatin meal is excreted in human urine as unhydrolyzed hydroxyproline peptide, 41 reflecting the appreciable absorption of intact peptides that can occur when they are composed of certain amino acids. In contrast to these findings for glycine and hydroxyproline peptides, recent studies in the rat with methionine and the diand tripeptides containing methionine have shown that absorption from the peptide is identical to that for the equivalent amino acid solution as long as physiological concentrations (50 mM amino acid equivalent or less) are used. 35 Furthermore, high concentrations of free methionine accumulate in the intestinal luminal contents during methionyl-methionyl-methionine absorption suggesting that hydrolysis of the tripeptide occurs at the intestinal brush border surface so rapidly that the methionine transport mechanism cannot accept all of the released amino acid. Hence, in . the case of methionine-containing peptides, hydrolysis of the peptide occurs very rapidly at the brush border and absorption is mainly, if not entirely, in the form of the amino acid. Schema for Intestinal Peptide Digestion and Absorption Based on the cellular location of the various peptidases and intubation experiments in man, both the brush border and intra-

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cellular peptidases appear to play a role in oligopeptide digestion, the site of hydrolysis depending on the types of amino acids and probably their location in small peptides. Transport mechanisms for amino acids are considered further below, but the recent protein meal studies in man 32· 33 strongly suggest that only the neutral amino acids and the two basic amino acids arginine and lysine are quantitatively taken in as amino acids. The imino acids (proline and hydroxyproline), glycine and the dicarboxylic acids (glutamic and aspartic) all appear to enter as constituents of small peptides, where specific intracellular peptides42 can hydrolyze them. Figure 4 diagrams the major mechanisms by which the intestinal cell appears to accommodate small peptides and amino acids. Whereas neutral peptides are hydrolyzed readily by brush border peptidases, particularly if a neutral amino acid is located at the amino-terminal position, some of these appear capable of entering the cell intact 43 and peptides containing mainly glycine residues or proline and hydroxyproline residues appear to enter the cell intact for lack of brush border peptidases; intracellular enzymes then reduce these to amino acids (90%) but approximately lO ~o of absorbed glycine or proline peptides leave the intestinal cell in peptide form (fig. 4). A mixed peptide containing different types of amino acids may utilize more than one entry mechanism depending on its relative affinity for specific cell transport or brush border hydrolytic systems. Arginine and lysine residues in protein become positioned on the C-terminus of peptides produced by trypsin and are split off by carboxypeptidase B to the free amino acids. Hence absorption of these dibasic amino acids probably does not occur m peptide form. Transport of Amino Acids Previous reviews •... 6 have carefully considered the various intestinal transport systems and table 1 briefly lists them along with their distinguishing characteristics. The neutral amino acid mechanism appears to be the functionally most important transport system and has been extensively

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PORTAL ~

IMINO

PEPTIDASE

90"

'

PROLINE

HYDROXY· PROLINE

--......,1---...

10'%

'-

SMALL )--.PEPTIDES--- - - -

/'lo"'

FIG. 4. Outline of the major routes of oligopeptide and amino acid entry into the intestinal cell. The peptides probably average three to four amino acid residues. Pro, proline; Gly , gylcine. TABLE Type

Neutral (monoaminomonocarboxylic)

Dibasic (diamino) Dicarboxylic (acidic)

Imino acids and glycine

a

1. Intestinal amino acid transport mechanisms Am ino ac ids t ransported

T ype of 1ransport

Aromatic (tyrosine, tryptophan, phenyl- Active, Na +-depenalanine dent Aliphatic (glycine, a alan ine, serine, threonine, valine, leucine, isoleucine) Methionine, histidine, glu tamine, a sparagine, cysteine Lysine, arginine, ornithine, cystine Active, partially Na +dependent Carrier-mediated, Glutamic acid, aspartic acid ?active, partially Na +-dependent Proline, hydroxyproline, glycine" Active, ?Na •-dependent

Relati ve rate

Very rapid

Rapid (10' i of neutral) Rapid

Slow

Shares both the neutral and imino mechanism with low affinity for the neutral.

studied. It is lmown to be an active process that will move the amino acid uphill against a cell to lumen concentration gradient. 47 Na + is required 48 • 49 and kinetic studies strongly suggest that a ternary complex of amino acid, Na +, and membrane carrier (presumably a protein) is formed thereby permitting entry and release beyond the outer cell barrier. 50 Na + then appears to be actively extruded from the cell to the lateral intercellular spaces thereby providing the driving force for so-called active amino acid transport. An intact carboxyl group

attached to the a-carbon is mandatory for active neutral amino acid transport and affinity is greatly increased when the aNH2 is unsubstituted and the side chain attached to the a-carbon is nonpolar (aliphatic). 47 Intestinal perfusion experiments in man have shown maximal rates of absorption in jejunum 51 as well as competition between glycine and alanine, 52 indicating that they share the neutral transport mechanism. Another study, using an equimolar mixture of eight neutral amino acids demonstrated relative absorption rates to

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be methionine > isoleucine > leucine > valine > phenylalanine > tryptophan > threonine, 53 corroborating the affinity requirements previously established in vitro in animals. The dibasic amino acids and cystine are actively absorbed by a Na + dependent process 54 that is distinct from the mechanism for neutral amino acids and operates at about 10% of the rate of the neutral system. 55 Unlike the neutral mechanism, it appears capable of transporting very slowly (10% of maximal) even in the absence of Na+. Proline, hydroxyproline, and glycine share a transport system that actively absorbs them against a concentration difference56 and may not have an absolute requirement for Na+. Glutamic and aspartic acid (the dicarboxylic amino acids) have recently been shown to be transported by a carrier mechanism that is partially Na + dependent. 57 Because these amino acids are rapidly removed by transamination by the intestine after uptake, it has not been possible to determine whether they are transported against a cell to lumen concentration gradient, but, considering the similarity to the neutral and basic mechanisms, active transport probably occurs. As noted above, the imino and dicarboxylic transport systems may not be of physiological importance since small peptides containing these amino acids are absorbed intact.

541

Interactions of Amino Acids and Hexoses:A Single Transport Mechanism? The actively absorbed sugars D-glucose and D-galactose have recently been shown to inhibit the transport of neutral amino acids 60· 61 prompting the suggestion that hexoses and amino acids may share a single carrier mechanism, 61 -63 a "polyfunctional" carrier 62 • 63 for which they must compete. However, if intestinal cells are allowed to accumulate leucine, addition of galactose to the lumen does not produce an exit of leucine from the cell into the lumen; that is, "countertransport" does not occur 64 as would have been expected if a single bidirectional carrier were handling both hexose and amino acid. Although the exact mechanism of interaction of glucose and amino acid is not known, it is likely that they are competing for the same, limited source of energy. 65

Absorption of Unhydrolyzed Protein It has been known for several years that the immature intestinal cell of the newborn animal continues to absorb intact proteins for several weeks after birth. 66 This process may assist the animal in assimilating maternal globulins in milk and has been assumed to occur by a passive mechanism. However, recent work in vitro has demonstrated that methionine in the medium greatly reduces the capacity of everted intestinal sacs from newborn piglets to absorb lactoglobulin. 67 Furthermore, uptake of -y-globulin reStimulation of Lysine Transport quires oxygen and Na + and is reversibly by Leucine inhibited by metabolic antagonists. 68 Leucine has recently been shown to aug- Thus, as more information unfolds, it apment the transport of lysine and arginine pears that even the absorption of unhyacross the intestinal cell. 58 · 59 This finding drolyzed globulins in the newborn may be is surprising since the basic and neutral · a complex and specific process. amino acid transport systems are known to The Future be distinct from one another. The interThe relative importance of brush border action appears to somehow augment the efflux of lysine from the serosal side of the and intracellular oligopeptidases in prointestinal cell by a mechanism that is not tein assimilation has not yet been precisely yet understood. 59 Such a special interre- defined and will depend heavily upon thorlationship of the neutral and basic amino ough study of the specificity of the individacids may serve a facilitating role during ual purified enzymes. Such information can protein absorption (fig. 4) . then be correlated with in vivo physiologi-

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cal experiments on the fate and interaction of small peptides and amino acids during digestion and transport.

Summary 1. The initial stage of digestion of dietary protein is accomplished by sequential action of activated pancreatic proteases within the intestinal lumen, yielding about 30% neutral and basic amino acids and 70% small peptides. 2. The amino acids are transported by their specific mechanisms. The oligopeptides are probably either hydrolyzed by brush border enzymes on the intestinal surface (particularly peptides containing mainly neutral amino acids) and the released amino acids transported, or they enter the intestinal cell intact (glycine, proline-hydroxyproline, dicarboxylic type peptides) to be hydrolyzed by soluble intracellular enzymes. 3. Approximately 10% of peptides containing predominantly glycine or prolinehydroxyproline enter and exit from the intestinal cell unhydrolyzed. REFERENCES 1. Northrop JH, Junitz M, Herriott RM: Crystalline Enzymes. Second edition. New York, Columbia University Press, 1948 2. Keith CA, Kazenko A, Laskowski M: Studies on proteolytic activity of crystalline protein B prepared from beef pancreas. J Bioi Chern 170:227238, 1947 3. Brown KD, Shupe RE, Laskowski M: Crystalline activated protein B (chymotrypsin B) . J Bioi Chern 173:99-107, 1948 4. Hartley BS: Amino acid sequence ofbovin chymo. trypsinogen. Nature (London) 201:1284-1287, 1964 5. Walsh KA, Kauffman DL, Sampath-Kumar KSV, et a!: On the structure and function of bovine trypsinogen and trypsin. Proc Nat Acad Sci USA 51:301-308, 1964 6. Shotton DM, Harley BS : Amino-acid sequence of porcine pancreatic elastase and its homologies with other serine proteinases. Nature (London) 225:802-806, 1970 7. Keller PJ : Pancreatic proteolytic enzymes, Handbook of Physiology, sect 5: Alimentary Canal. Edited by CF Code. Washington DC, American Physiological Society, 1968, p 2605-2628

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8. Nordstrom C, Dahlqvist A: The cellular localization of enterokinase. Biochim Biophys Acta 198: 621-622, 1970 9. Jacobsen CF, Leonis J: A recording auto-titration. Compt Rend Trav Lab Carlsberg, Ser Chim 27:333-339, 1951 10. Dreyer WJ, Neurath H: The activation of chymotrypsinogen. J Bioi Chern 217:527-539, 1955 11. Keller PJ, Cohen E, Neurath H: Procarboxypeptidase. II. Chromatographic isolation, further characterization, and activation. J Bioi Chern 230:905-915, 1958 12. Cox DJ, Wintersberger E, Neurath H : Bovine pancreatic procarboxypeptidase B. II. Mechanism of activation. Biochemistry (Wash) 1:1078-1082, 1962 13. Sanger F, Thppy H : The amino-acid sequence in the phenylalanyl chain of insulin. 2. The investigation of peptides from enzymic hydrolysates. Biochem J 49:481-490, 1951 14. Gladner JA, Folk JE: Carboxypeptidase B. II. Mode of action on protein substrates and its application to carboxyl terminal group analysis. J Bioi Chern 231:393-401, 1958 15. Folk JE, Cole PW: Chymotrypsin C. II. Enzymatic specificity toward several polypeptides. J Bioi Chern 240:193-197, 1965 16. Naughton MA, Sanger F: Purification and specificity of pancreatic elastase. Biochem J 78:156163, 1961 17. Josefsson L, Lindberg T , Ojesjo L: Intestinal dipeptidases. Dipeptidase activities in human intestinal juice. Scand J Gastroent 3:207-210, 1968 18. Robinson GB, Shaw B: The hydrolysis of dipeptides by different regions of rat small intestine. Biochem J 77:351-356, 1960 19. Lindberg T: Intestinal dipeptidases. Dipeptidase activity in the mucosa of the gastrointestinal tract of the adult human. Acta Physiol Scand 66:437443, 1966 20. Rhodes JB, Eichholz A, Crane RK: Studies on the organization of the brush border in intestinal epithelial cells. IV. Aminopeptidase activity in microvillus membranes of hamster intestinal brush borders. Biochim Biophys Acta 135:959965, 1967 21. Heizer WD, Isselbacher KJ: Intestinal peptide hydrolases: Differences between brush border and cytoplasmic enzymes. Clin Res 18:382, 1970 22. Kim YS, Birtwhistle W, Kim YW, eta!.: Intestinal peptide hydrolases: multiple forms of enzymes in cytoplasm and brush border (abstr). Gastroenterology 60:685, 1971 23. Josefsson L, Noren 0, Sjostrom H: Comparison of dipeptidase activity in different tissues of the pig. Acta Physiol Scand 72:108-114, 1968

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