Some physical and immunological properties of ox kidney biliverdin reductase.

The liver, kidney and spleen of the mouse and rat and the kidney and spleen of the ox express a monomeric form of biliverdin reductase (Mr 34,000), which in the case of the ox kidney enzyme exists in two forms (pI 5.4 and 5.2) that are probably charge isomers. The livers of the mouse and rats express, in addition, a protein (Mr 46,000) that cross-reacts with antibodies raised against the ox kidney enzyme and may be related to form 2 described by Frydman, Tomaro, Awruch & Frydman [(1983) Biochim. Biophys. Acta 759, 257-263]. Higher-Mr forms appear to exist in the guinea pig and hamster. The ox kidney enzyme has three thiol groups, of which two are accessible to 5,5'-dithiobis-(2-nitrobenzoate) in the native enzyme. Immunocytochemical analysis reveals that biliverdin reductase is localized in proximal tubules of the inner cortex of the rat kidney. Biliverdin reductase antiserum also stains proximal tubules in human and ox kidney. The staining of podocytes in glomeruli of ox kidney with antiserum to aldose reductase is particularly prominent. The localization of biliverdin reductase in the inner cortical zone of rat kidney is similar to that described for glutathione S-transferase YfYf, and it is suggested that one function of this 'intracellular binding protein' may be to maintain a low free concentration of biliverdin to allow biliverdin reductase to operate efficiently.     

Characterization of ferrochelatase in kidney and erythroleukemia cells

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

Utilization of starch and synthesis of a combined amylase/αα-glucosidase by the human colonic anaerobe Bacteroides ovatus

Bacteroides ovatus preferentially utilized starch and pectin when grown on a mixture of polysaccharides in batch culture, indicating that these carbohydrates are important substrates for the bacterium in the human large intestine. Further studies on starch breakdown showed that continuous cultures grew on the polysaccharide when it provided the sole carbohydrate source, to yield a single hydrolytic product at low dilution rates ( D = 0·04 h⁻¹), with an estimated molecular mass of 13 kDa. In contrast, two major types of oligomeric products were formed at higher dilution rates ( D = 0·44 h⁻¹), with approximate molecular weights of 11 and 140 kDa. Analysis of cell-associated starch-degrading enzymes produced by Bact. ovatus using ion exchange chromatography and HPLC gel-filtration showed that amylase and α-glucosidase activities eluted in the same fractions. The single peak containing amylase and α-glucosidase activities obtained by HPLC gel-filtration chromatography corresponded to a molecular mass of approximately 140 kDa, and activity staining of gels for α-glucosidase activity after polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate, gave an estimated molecular mass of 70 kDa, indicating this enzyme to be a dimer. After renaturation, the 70 kDa band was cut from the gels and solubilized. The extract hydrolysed gelatinized starch and p -nitrophenyl-α- D -glucopyranoside.     

Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells

Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.     

Aspartate aminotransferase from Panicum maximum Jacq. var. trichoglume Eyles, a C4 plant: purification, molecular properties, and preparation of antibody

Extracts of the leaf tissue of Panicum maximum Jacq. var. trichoglume Eyles (a phosphoenolpyruvate carboxykinase type of C4 plant) were examined and at least two isoforms of aspartate aminotransferase (EC 2.6.1.1), with different electrophoretic mobilities, were detected. The predominant isoform was purified to homogeneity from mesophyll cells. The purification procedure included fractionation with ammonium sulfate followed by chromatography on diethylaminoethyl-cellulose, Sephacryl S-300, and hydroxyapatite. The purified enzyme had specific activities of 182 and 165 mumol/min/mg protein, measured in terms of the synthesis of oxaloacetate and aspartate, respectively, at pH 8.0. The enzyme, with an apparent molecular size of 100 kDa, appears to be a dimer of a single polypeptide with a molecular size of 42 kDa. Mono specific polyclonal antibodies were raised against the 42-kDa polypeptide. Only a single stained band was detected in extracts of whole leaves by immunoblot analysis with this antibody after two-dimensional polyacrylamide electrophoresis. Furthermore, no difference in mobility was observed between the enzymes extracted from mesophyll and bundle sheath cells on native polyacrylamide gels. These findings are discussed in relation to the other isoform in the leaves of this species.     

Purification and characterization of D-3-aminoisobutyrate-pyruvate aminotransferase from rat liver

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) was purified 1900-fold from rat liver extract. The purified enzyme showed a molecular mass of 180 kDa by gel-permeation HPLC analysis using a TSK gel G3000SW column. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate resulted in identification of a single band of approx. 50 kDa, indicating that the native enzyme is probably a tetrametric protein. The specific activity of the purified enzyme was 1.14 mumol/min per mg protein. D-3-Aminoisobutyrate and beta-alanine were good amino donors. The Km value for L-3-aminoisobutyrate was 100-times larger than that for the D-isomer. The apparent Km values for D-3-aminoisobutyrate and beta-alanine were 35 and 282 microM, respectively. Pyruvate, glyoxylate, oxalacetate, 2-oxo-n-valerate, and 2-oxo-n-butyrate were good amino acceptors. The apparent Km values for pyruvate and glyoxylate were 32 and 44 microM, respectively.     

Characterization of two different alkaline phosphatases in mouse teratoma: Partial purification,electrophoretic, and histochemical studies

Alkaline phosphatase (E.C.3.1.3.1.) has been used as a marker for embryonal carcinoma cells which constitute the multipotential stem cells of the mouse teratoma. Studies by other investigators based on kinetics of thermal inactivation and L-phenylalanine inhibition have shown that the alkaline phosphatase of the teratoma differs from the mouse intestinal and liver isozymes, but resembles the isozymes of kidney and placenta. Since functional characterization of nonpurified enzymes is not the most accurate means for distinguishing different molecular forms of an enzyme, we have partially purified the enzymes from the ascitic (embryoid body) and solid tumor forms of the OTT-6050 teratoma line, and utilized the technique of electrophoresis in polyacrylamide gels to compare the teratoma enzyme with isozymes from kidney and placenta. Covalent ³²PO₄-labeling of the alkaline phosphatases and polyacrylamide gel electrophoresis in sodium dodecylsulfate was also used to compare the subunit molecular weights of the enzymes. The results indicate that the mouse teratoma enzyme is distinct from the kidney and placental isozymes. Since histochemical studies have localized the enzyme to the stem cell population of the teratoma, the results imply that stem cell alkaline phosphatase is a distinct isozyme. The embryoid bodies contain a second alkaline phosphatase which may correspond to the placental isozyme. This enzyme may be attributed to the outer cell layer of embryoid bodies of the ascitic tumor, since this cell type histochemically demonstrates alkaline phosphatase activity.     

Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD⁺ dependent enzyme which was inactivated by trypsing and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.     

Purification, characterization and subcellular localization of pig liver alpha-L-iduronidase

alpha-L-Iduronidase was purified about 100,000-fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A-Sepharose 4B, heparin-Sepharose 4B, Toyopearl HW-55, Sephadex G-100 and chelating Sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G-100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium dodecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70-kDa and 62-kDa components were confirmed as alpha-L-iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0-3.5 and 65 microM 4-methylumbelliferyl-alpha-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5-6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.     

Further characterization of a novel phospholipase B (phospholipase A2--lysophospholipase) from intestinal brush-border membranes.

The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 - lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate - polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5-10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5-10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.     

Biliverdin reductase: substrate specificity and kinetics

The substrate specificity of the different forms of rat liver biliverdin reductase was examined using synthetic biliverdins. Biliverdins carrying methyl, ethyl and one propionate residue in their structure were not substrates of biliverdin reductase. Biliverdins with one propionate and one acetate residue or with two acetate residues were not reduced by the enzyme either. The presence of two propionates in the biliverdin structure gave a biliverdin with substrate activity. Increasing the number of propionates to four, as in coprobiliverdins, did not affect substrate activity, while the octaacid urobiliverdins were also good substrates of the enzymes. The beta isomer of urobiliverdin III and coprobiliverdin III were reduced at much higher rates by molecular form 3 of the enzyme as compared to molecular form 1, a fact which had already been observed with the beta isomer of biliverdins IX, XIII and hematobiliverdin. All the biliverdins mentioned above were readily reduced to bilirubins by sodium borohydride. The purified molecular forms 1 and 3 displayed sigmoidal kinetics with most of the biliverdins tested. The data were analyzed by nonlinear regression in a microcomputer and it was found that they fitted a model of a moderate cooperative dimer where both ES and ES2 are catalytically active. The Vm, Ks and the Hill numbers, nH, for biliverdin IX alpha and beta, hematobiliverdin IX alpha and beta, and several synthetic biliverdin isomers are given. Molecular form 2 showed classical Michaelian kinetics.     

Electrophoretic mobility of gamma-glutamyltransferase in rat liver subcellular fractions.Evidence for structure difference from the kidney enzyme.

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.     

Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization

It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa. All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE.     

α-1,4-Glucan phosphorylase forms in the green alga Eremosphaera viridis

In extracts of the unicellular green alga Eremosphaera viridis DeBary (Chlorococcales, Chlorophyceae) the average specific activity of α-1,4-glucan phosphorylase (E.C. 2.4.1.1) was 200 nmol glucose 1-phosphate formed per min and mg protein. Using continuous and discontinuous electrophoresis on polyacrylamide gels, three phosphorylase forms were found. When the log of the relative mobility of the three enzyme forms was plotted versus the acrylamide gel concentration (Ferguson plot) parallel lines were obtained, indicating that the three enzymes were indiscernible with respect to molecular weight. Electrophoresis on density gradient gels resulted in three activity zones lying close to each other. The relative molecular mass ( M _r) of the three enzymes was estimated to be around 180,000 with a difference of less than 7,000 between the small and the large forms.     

Identification of the amino acid residues essential for the activity and the interconversion of the molecular forms of biliverdin reductase

Biliverdin reductase (molecular form 1, EC 1.3.1.24, bilirubin:NAD(P)+ oxidoreductase) carries three thiol residues. Only one of them could be alkylated when a ratio N-ethylmaleimide (NEM)/mol enzyme's SH = 90 was used. The alkylation of this thiol group inhibited the conversion of molecular form 1 to its dimer, molecular form 3; however, it did not inhibit the enzymatic activity. At a ratio of NEM/enzyme's SH = 300, two thiol residues were alkylated and the activity of the enzyme was totally inhibited. The third thiol group could not be alkylated either by NEM or by iodoacetamide. Biliverdin as well as the co-substrate NADPH protected the thiol residue essential for the enzymatic activity from alkylation. Spectroscopic evidence was obtained that this thiol group binds covalently to the C-10 of biliverdin to form a rubinoid adduct. The presence of a lysine residue, which is also essential for the enzymatic activity, could be inferred from the fact that by reduction of the Schiff base formed by the enzyme with pyridoxal phosphate the catalytic activity was irreversibly abolished. The location of a lysine residue in the vicinity of the thiol group involved in the catalytic activity was evident when the enzyme was treated with o-phthalaldehyde. The inactivation of the enzymatic activity was coincident with the formation of the fluorescent isoindole derivative which originates when the thiol and epsilon-NH2 groups are located about 3 A apart. The presence of a positively charged ammonium ion in the vicinity of the NADPH binding site was inferred from the shifts in the UVmax of NADPH from 340 nm to 327 nm and of 3-acetyl NADPH from 360 nm to 348 nm when the pyridine nucleotides bind to the reductase. The involvement of arginine residues in the enzymatic activity was established by inhibition of the latter after reaction with butanedione. This inhibition was totally protected by NADPH but not by biliverdin. The similarity of the structural features of biliverdin reductase with those of several dehydrogenases is discussed.     

Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis.

A modified method for the detection of DNA polymerases in cell extracts and purified enzyme preparations after electrophoresis in polyacrylamide gradient cylindrical gels is described. The technique, which is based on direct assay of activity in a reaction mixture during elution of DNA polymerases from gel slices, was applied to the pursuit of enzyme forms of Streptomyces aureofaciens DNA polymerase during purification procedure. In a crude extract of S. aureofaciens mycelium many catalytically active forms of DNA polymerase ranging from 35 to 860 kDa were detected. After purification, that included mycelium homogenization, precipitation of nucleic acids by polyethylene glycol, DEAE-Sephadex, QAE-Sephadex and DNA-Sepharose chromatography, higher molecular mass forms of more than 172 kDa have not been found. The lower molecular mass forms were separated into two groups by DNA-Sepharose chromatography. On the basis of their characterization, it is assumed that the lower molecular mass forms are produced by proteolysis and the major form found after purification of S. aureofaciens DNA polymerase in the presence of suitable proteinase inhibitors should be a protein of 172 kDa.     

A study of the heterogenous structure of guinea pig lysosomal beta-mannosidase using a polyclonal antibody

Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.     

Proteinases in molting fluid of the tobacco hornworm,Manduca sexta

Manduca sexta pharate pupal molting fluid contains more than 10 proteolytic enzymes that differ in relative mobility during electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate and gelatin. The major gelatin digesting enzyme was an endoprotease with an apparent molecular weight of 100 kDa. Gel filtration on a Sephacryl S-300 column resolved another endoprotease of similar size that digests azocoll and [³H]casein. In addition we found an aminopeptidase-like enzyme (MW_app 500 kDa) and at least three carboxypeptidase-like enzymes (MW_app 10–60 kDa). Use of pseudosubstrates and inhibitors suggested the presence of both trypsin-like and chymotrypsin-like enzymes with the former activity approx. 10-fold greater than the latter. However, none of the proteolytic enzymes were substantially inhibited by diisopropylphosphorofluoridate or phenylmethylsulfonyl fluoride which are poteint inhibitors of trypsin and chymotrypsin. No carboxyl or sulfhydryl proteases were detected. The enzymes were most active in the neutral to alkaline pH range, but they were relatively unstable during storage which precluded their purification to homogeneity. Proteolysis of Manduca cuticular protein appears to involve a rather complex and unique mixture of endo- and exo-cleaving proteolytic enzymes.