Cleavage of Rabbit Myelin Basic Protein by Pepsin

Rapid cleavage of bovine and guinea pig myelin basic proteins by pepsin at pH 6.0 is limited to the Phe-Phe bond in the middle of the molecule. In the rabbit protein, however, rapid cleavages occur elsewhere in addition to the Phe⁸⁷-Phe⁸⁸ bond in regions in which there are amino acid substitutions. Rapid cleavage occurs at the Leu¹⁵¹-Phe¹⁵² bond, at which Ile-151 has been replaced by Leu, the residue that actually contributes the scissile bond. Rapid cleavages occur at the Phe⁴⁴-Phe⁴⁵ and Leu¹⁰⁹-Ser¹¹⁰ bonds, which in the bovine and guinea pig proteins are relatively resistant under the experimental conditions (pH 6.0). The increased susceptibility of these bonds in the rabbit protein appears to be related to the replacement of Gly-46 by Ser and the change in the sequence immediately NH₂-terminal to Leu-109, from Leu-Ser to Thr-Val. These cleavages of the rabbit protein at the four very susceptible bonds have permitted us to isolate peptides (1-44), (45-87), (88-109), (110-151), and (152-168) in high yield. We have also isolated peptides (88-151), (1-14), and (15-44) in low yield; the latter two result from limited cleavage at the relatively resistant Tyr¹⁴Leu¹⁵ bond. Peptide (88-109) has been chromatographically resolved into species differing in the degree of methylation of Arg-105; this resolution is thought to result from differences in hydrogen bonding ability of the guanidinium groups.     

Isolation and Identification of Large Overlapping Fragments of Rabbit Myelin Basic Protein Produced by Limited Peptic Hydrolysis

Treatment of rabbit myelin basic protein component 1 with pepsin (enzyme:substrate, 1:500 w/w) in 0.5 M-ammonium formate (pH 6.00) for 15-20 min at room temperature resulted in limited cleavage of the protein. The resulting fragments were isolated by ion-exchange chromatography and gel filtration and identified by amino acid and COOH-terminal analyses and by tryptic peptide mapping. All of the possible products resulting from incomplete cleavages at the highly susceptible Phe44-Phe45, Phe87-Phe88, Leu109-Ser110, and Leu151-Phe152 bonds were isolated: peptides (1-151), (1-109), (1-87), (45-168), (45-151), (45-109), (88-168), (88-151), and (110-168). Of these, peptides (1-151), (1-87), and (88-151) were recovered in the greatest yield (0.14-0.19 mol per mol of starting protein). Relatively low yields (0.04 mol/mol starting protein) were obtained for peptides (1-109) and (110-168), indicating that the Leu109-Ser110 bond is somewhat more resistant to peptic cleavage than are the Phe-Phe and Leu-Phe bonds. Smaller fragments of the basic protein were also recovered: peptides (1-44), (1-28), (45-87), (88-109), (110-151), and (152-168). Many of the individual peptides could be readily identified in electrophoretograms of the total peptic digest. The relative electrophoretic mobilities of the above-mentioned peptides, together with the previously isolated peptides (1-14) and (15-44), were determined in 15% (w/w) polyacrylamide slab gels containing 1 M-acetic acid and 8 M-urea.     

Cleavage of Rabbit Myelin Basic Protein by Thrombin

Rabbit myelin basic protein (BP) contains several Arg-X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin-to-substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95)-Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme-to-substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)-Phe(44) and Arg(128)-Ala(129) bonds, but with some cleavage at the Arg(31)-His(32) and Arg(63)-Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96-168), which we examined in detail, nearly complete cleavage of the Arg(128)-Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds. The susceptibilities to cleavage of the Arg-X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)-Ala(129) and Arg(160)-Ser(161), suggests the occurrence of a chain reversal or beta-turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe-Phe(87-88) sequence is left intact.     

The influence of pH on the degradation of bovine myelin basic protein by bovine brain cathepsin

The degradation of bovine myelin basic protein by bovine brain cathepsin D (ED 3.4.23.5) was studied over a pH range of 2.75 - 6.0. Throughout this pH range pepstatin, an inhibitor of cathepsin D, prevented the degradation. The degradation at a pH away from the optimum of pH 3.5 was predictably slower, but also resulted in more restricted cleavage. Above pH 4.5 bovine basic protein peptide 1 - 42 was not degraded further to peptide 1 - 36 as occurs at pH 3.5. Additionally, at pH 5.5 another fragment of basic protein, peptide 1 - 91, persisted indicating that under certain basic protein as well as basic protein peptide 43 - 169 may be cleaved in the molecular region of basic protein around the phenylalanyl-phenylalanine residues at position 88 - 89. The small amount of peptides 1 - 91 and 92 - 169 detected at pH 5.5 suggests that the bond between residues 91 and 92 in intact basic protein is a minor cleavage site. The options and variation in cleavage around residues 88 - 92 of basic protein presumably result from pH-dependent changes in conformation in the is region but could also be due to changes in conformation of cathepsin D. These results indicate that local tissue changes such a pH amy affect not only the velocity of the reaction but also the nature of th product formed by the degradation of basic protein by brain cathepsin D     

Treatment of an encephalitogenic peptide from guinea pig myelin basic protein with alpha-protease and thermolysin. Isolation of fragments and determination of cleavage sites.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.     

Experimental allergic encephalomyelitis in the Lewis rat: farther delineation of active sites in guinea pig and bovine myelin basic proteins.

Highly encephalitogenic peptide (37-88), derived from the guinea pig myelin basic protein by peptic digestion, was treated chemically to destroy its tyrosyl and histidyl residues and enzymatically to remove its C-terminal sequence Val-His-Phe. Neither of the modifications resulted in loss of activity in Lewis rats. The enccephalitogenic region within peptide (37-88) was located by examination of derivative peptides obtained by selective proteolytic cleavage. The results showed that peptide (61-88), like peptide (43-88), was fully active at the level of 0.02 nmole whereas peptides (72-88) and (72-84) were fully active at levels of 0.5 and 2.5 nmole, respectively. In contrast, peptides (43-71) and (75-88) were completely inactive. These results demonstrated that the undecapeptide Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro (residues 72-84), although not as encephalitogenic as peptides (43-88) or (61-88), does contain the elements essential for the induction of disease. At the levels tested (10.8 and 2.2 nmole) only peptides (43-88) and (61-88) were capable of inhibiting the induciton of disease by passively transferred lymph node cells; this inhibition, however, was less than that achieved by the intact guinea pig basic protein. Further studies on the encephalitogenicity of the bovine basic protein in Lewis rats demonstrated that the active site in the C-terminal half of this protein is present in its entirety within residues 89 to 115.     

Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.     

Limited Digestion of Guinea Pig Myelin Basic Protein and Its Carboxy-Terminal Fragment (Residues 89–169) with Staphylococcus aureus V8 Protease

Staphylococcus aureus V8 protease has been reported to have a strict specificity for cleavage of the Glu-X bond in ammonium bicarbonate (pH 7.9). With myelin basic protein and one of its major peptic fragments (residues 89-169) as substrates, selective cleavage of Asp(32)-Thr(33), Asp(37)-Ser(38), and Glu(118-Gly(119) bonds was observed, as well as the unusual cleavage of the Gly(127)-Gly(128) bond. The Asp-Glu and Glu-Asn bonds in the sequence of Gln-Asp-Glu-Asn-Pro(81-84) were resistant to V8 protease attack. The following peptides were identified as products of limited cleavage of basic protein by V8 protease: (1-32), (1-37), (33-169), (38-169), (33-118), (38-118), (33-127), (38-127), (119-169), and (128-169). Cleavage of the peptic peptide (89-169) yielded fragments (89-118), (89-127), (119-169), and (128-169). All peptides were identified by amino acid analysis, as well as NH2- and COOH-terminal analyses. Time course studies with basic protein showed that V8 protease initially attacked the bonds between Asp(32) and Thr(33) and Asp(37) and Ser(38). With peptide (89-169) the initial cleavage was between Glu(118) and Gly(119). Peptides (89-118) and (89-127) were encephalitogenic in the Lewis rat. The activity of these peptides in the rat confirms the presence of a minor encephalitogenic site in guinea pig basic protein. Peptide (89-127) was encephalitogenic in the guinea pig, as expected, because it contains the intact encephalitogenic site. V8 protease digestion of basic protein yields some interesting new fragments, not previously available for biologic studies.     

Aspergillus oryzae acid proteinase. Purification and properties, and formation of pi-chymotrypsin.

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.     

Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies

Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP.     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.     

Sequence of Guinea Pig Myelin Basic Protein

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.     

Guinea pig MSEL-neurophysin. Sequence comparison of eight mammalian MSEL-neurophysins

The amino acid sequence of guinea pig MSEL-neurophysin has been determined using tryptic peptides derived from the performic acid-oxidized protein and staphylococcal proteinase peptides obtained from the reduced-carboxamidomethylated neurophysin. Guinea pig MSEL-neurophysin consists of a 93-residue polypeptide chain that shows 12 substitutions and 2 deletions when compared to bovine MSEL-neurophysin. It displays the highest number of variations among known mammalian MSEL-neurophysins. These variations are mainly found in the C-terminal region (residues 88-93). Moreover guinea pig MSEL-neurophysin, like rat homologous protein, exhibits substitutions in positions 2, 5, 29 and 81 and lacks an arginine in the penultimate position. Comparison between eight mammalian MSEL-neurophysins reveals a highly conserved region (residues 1 to 88) and a hypervariable region (residues 89 to 93/95). On the other hand the eight species examined are endowed with arginine vasopressin except pig, which has a lysine vasopressin. In the vasopressin-MSEL-neurophysin precursor, the hormonal moiety and the MSEL region of neurophysin (residues 1-9) are encoded by a common exon in ox, rat and man; it can be concluded that this exon is evolutionarily conservative in contrast to the one encoding the C-terminal region of MSEL-neurophysin.     

COMPARATIVE STUDIES OF GUINEA PIG AND BOVINE MYELIN BASIC PROTEINS. PARTIAL CHARACTERIZATION OF CHEMICALLY DERIVED FRAGMENTS AND THEIR ENCEPHALITOGENIC ACTIVITIES IN LEWIS RATS

Guinea pig and bovine myelin basic proteins were chemically cleaved at the carboxyl peptide bonds of methionyl and tryptophanyl residues to yield several fragments. Comparison of the bovine fragment consisting of the first 20 residues of the protein with the corresponding guinea pig fragment showed that the latter differs in containing histidine and glycine (one residue of each), an additional threonyl residue, and one fewer alanyl residues. Comparison of the bovine fragment consisting of the C-terminal 54 residues of the protein (residues 117-170) with the corresponding guinea pig fragment showed that the latter differs in containing one fewer histidyl and leucyl residues and an additional phenylalanyl residue. Tests of encephalitogenic activity in Lewis rats showed that these two fragments from both species were much less active, on a molar basis, than the uncleaved protein. On the other hand, examination of the bovine fragments consisting of residues 1-116 and 21-116 and the corresponding fragments obtained from the guinea pig protein revealed activity at least as high as that of the respective uncleaved proteins.     

Cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from mouse and guinea pig liver Golgi membranes during the acute phase response

1. Rat Gal beta 1-4GlcNAc alpha 2-6sialyltransferase (E.C. 2.4.99.1) is released from Golgi membranes by cleavage of a portion of the enzyme containing the active site from a membrane anchor; this effect was most dramatic during the acute phase response. The enzyme that cleaved sialyltransferase had the properties of cathepsin D was most active at pH 5.6 and was likely of lysosomal origin (Lammers and Jamieson, 1988). 2. The acute phase response of sialyltransferase in mouse and guinea pig was previously found to differ from that in the rat. Release of sialyltransferase from mouse and guinea pig Golgi membranes has now been studied in order to make a comparison with the rat system. 3. Maximum release of sialyltransferase from mouse and guinea pig Golgi occurred at pH 4.6 and 5.2, respectively; like the rat a cathepsin D-like proteinase was responsible for release of both enzymes. 4. Immunoblot analysis showed that membrane-bound rat and mouse sialyltransferase had Mr 49,000, whereas the guinea pig enzyme had Mr 42,000. The released form of the rat enzyme had Mr 42,000, but released forms of mouse and guinea pig enzymes had Mr 38,000 suggesting a different cleavage site for these two enzymes compared to the rat enzyme.     

Evidence that differentiates between precursor cleavages at dibasic and Arg-X-Lys/Arg-Arg sites.

It is well known that precursor cleavage at paired basic amino acids (e.g., Lys-Arg, Arg-Arg) within the regulated secretory pathway is one of the key steps to produce bioactive peptides. On the other hand, we have recently shown that precursors with an Arg residue at the fourth residue upstream of the cleavage site besides the basic pair, i.e. with the Arg-X-Lys/Arg-Arg (RXK/RR) motif, are cleaved within the constitutive secretory pathway. To discriminate between the precursor cleavage at RXK/RR sites within the constitutive pathway and that at dibasic sites within the regulated pathway, we examined the effects of drugs affecting the secretory process, intracellular Ca2+ depletion, and a protease inhibitor on these cleavages. Chloroquine (a weak base), depletion of intracellular Ca2+ by A23187 (a Ca2+ ionophore), and the Pittsburgh-type mutant of alpha 1-protease inhibitor differentially affected these two cleavages. Brefeldin A, which impedes protein transport from the endoplasmic reticulum to the Golgi complex, inhibited both cleavages. Colchicine (an anti-microtubular drug) had no discernible effect on either cleavage. These observations support the notion that the precursor cleavages at dibasic and RXK/RR sites occur in different subcellular compartments, and are catalyzed by different processing endoproteases.     

Synthetic peptides as substrates for processing enzymes in the bovine pituitary

Synthetic peptides, based on sequences of proopiomelanocortin (POMC) cleaved in both the bovine anterior and intermediate pituitaries (-Phe-Pro-Leu-Gly-Phe-Lys-Arg-Glu-Leu-Thr-Gly-) and only in the intermediate lobe (-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-), were used as substrates for the enzymes that process POMC to active hormones in the anterior and intermediate lobes of the pituitary. Cleavage of these peptides at the dibasic pair of residues, the expected cleavage site, was observed with a lysate from bovine pituitary secretory granules. Cleavage occurred optimally at a pH between 4 and 5 and was inhibited with sulfhydryl reagents, pepstatin, and leupeptin. Little specificity for the nature of the basic residues at the cleavage site was observed. An additional cleavage, following glutamic acid residues, was also seen.     

Metalloendoprotease Cleavage of 18.2- and 14.1-Kilodalton Basic Proteins Dissociating from Rodent Myelin Membranes Generates 10.0- and 5.9-Kilodalton G-Terminal Fragments

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37 degrees C; pH 7.4). After 1-3 h, electrophoresis of the membranes revealed loss of 50% of 18.2- and 14.1-kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane-associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4-kDa and minor 10.0- and 5.9-kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2- and 14.1-kDa forms of MBP were cleaved near residue 73 to produce a 10.0- and 5.9-kDa C-terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5-20% using leupeptin (20 microM) but up to 50% by dithiothreitol mM), phenylmethylsulphonyl fluoride (1 mM), and phosphoramidon (50 microM) but up to 50% by dithiothreitol (DDT, 10 mM). Only DDT and 1,10-phenanthroline substantially blocked the formation of the characteristic 10.0-and 5.9-kDa C-terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N-ethylmaleimide/leupeptin-sensitive calpains and phosphoramidon-sensitive endopeptidase 24.11.     

Enzymic degradation of the Fc fragment of rabbit immunoglobulin IgG

The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.