Enzymic solubilization of the human intestinal brush border membrane enzymes

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, γ-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush border membrane vesicles by various enzymes (especially pancreatic proteases) have been studied.The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases.Electron microscopy of brush border membrane vesicles demonstrates “knob-like” structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of th enzymic activities with the removal of the particles.The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

Differential sensitivity of intestinal brush border enzymes to pancreatic and lysosomal proteases.

Isolated human intestinal brush border membranes were used as sources of enzyme to study their degradation by proteolytic enzymes. Human intestinal brush border hydrolases undergo degradation by two separate proteolytic systems. Sucrase and alkaline phosphatase are degraded by pancreatic proteases (e.g. chymotrypsin) at neutral pH, whereas trehalase is degraded by lysosomal extracts at acid pH. Both the membrane bound and membrane free isolated enzymes had similar sensitivity to proteolytic enzymes. Thus, initial removal from the membrane is not essential as a prerequisite to proteolysis. It is postulated that the brush border membrane of the intestine is subject to proteolysis by pancreatic enzymes from the external cell surface and by lysosomal proteases within the cell.     

Phlorizin receptors in isolated kidney brush border membranes Differential enzymatic modification of high-affinity receptors and unspecific binding sites

High-affinity phlorizin receptors in isolated kidney brush border membranes are destroyed by the proteolytic enzymes trypsin and papain. The digested membranes show increased unspecific phlorizin binding. It is proposed, that both enzymes expose a deeper, more hydrophobic layer in the brush border membrane to explain the latter finding.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium

The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6–9.8, similar substrate k_m's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000–150,000, and both enzymes show an R_f value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The V_max of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in V_max are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Ca2+ inhibition of brush border alkaline phosphatase activity in Hymenolepis diminuta

The brush border membrane of Hymenolepis diminuta contains several Ca2+-dependent enzymes. Following our isolation of a Ca2+-dependent modulator protein we examined the kinetic properties of the brush border marker alkaline phosphatase from fractionated and crude tegument. We show that this enzyme is inhibited by Ca2+ concentrations approaching those in the calcareous corpuscles of H. diminuta.     

Immobilization of enzymes on chitin and chitosan

During the last few years, d-glucose isomerase, glucoamylase, β-d-galactosidase (lactase), β-d-glucosidase, d-glucose oxidase, AMP deaminase, urease, pronase, subtilisin, trypsin, papain, alkaline phosphatase, acid phosphatase, pepsin, chymotrypsin and lysozyme have been immobilized on chitin and on some of its derivatives, mainly with glutaraldehyde. The preparation and performances of the immobilized enzymes are described.     

Influence of duodenal secretions and its components on release and activities of human brush-border enzymes

The in vitro effects of human duodenal secretions and various combinations of its components on activity and release of enzymes from the human brush border were examined. Sucrase retained activity for 90 min in duodenal secretions, and maltase was almost as stable; lactase lost activity rapidly and alkaline phosphatase was of intermediate stability. Inactivation of lactase could only be partly (50%) attributed to luminal proteases, bile salts and phospholipids played no role. Rate of release of an enzyme from the brush border bore no relationship to its rate of inactivation. When individual proteases were studied, elastase was the most potent for releasing disaccharidases from the brush border; trypsin was ineffective alone but augmented the effect of elastase. Sucrase and maltase were activated by proteolytic release, but activation was abolished by simultaneous exposure of brush borders to bile salts. Lactase was released and rapidly inactivated by proteinases, while alkaline phosphatase appeared to be inactivated without significant release. These results show that there are significant interactions between luminal factors which have been inapparent when studying them in isolation. Loss of functionally useful enzyme does not follow release of sucrase or maltase from the brush border into the lumen but does follow release of lactase. Study of the susceptibility of lactase to inactivation by luminal factors in the various forms of lactose intolerance is warranted.     

Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.     

Studies on the characterization of the Rho(D) antigen.

The Rho(D) antigen of red cell membranes was solubilized using ethylene-diamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, and pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rho(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rho(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rho(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rho(D) molecule in its natural environment. The solubilized Rho(D) molecules are apparently not dependent on lipid for their Rho(D) activity.     

Acid phosphatase activity in the isolated brush border membrane of the tapeworm, Hymenolepis diminuta: partial characterization and differentiation from the alkaline phosphatase activity

The isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzes p-nitrophenyl phosphate over a broad pH range. Acid phosphatase activity (pH optimum at 4.0) is inhibited specifically by sodium dodecyl sulfate (SDS) and NaF, while the alkaline phosphatase activity (pH optimum at 8.8) is inhibited specifically by levamisole, 2-mercaptoethanol, and ethylenediaminetetra-acetate (EDTA). These two phosphatase activities are further differentiated in that (1) there is a rapid decrease in alkaline phosphatase activity when the membrane preparation is incubated at pH 4.0, while there is little loss of acid phosphatase activity, and (2) the alkaline phosphatase activity is solubilized with no loss of activity when the membrane is treated with Triton X-100, while such treatment causes a significant loss of acid phosphatase activity. Both activities are nonspecific and hydrolyze a variety of phosphorylated compounds, but the relative activities of the two phosphatases against these substrates vary significantly.     

Limited proteolysis of the catalytic subunit of cAMP-dependent protein kinase — A membranal regulatory device?

Brush border membranes isolated from rat small intestine were found to possess a cAMP-dependent protein kinase activity. Upon addition of cAMP, a rapid, time-dependent inactivation of this enzyme occurs, which was found to be due to a proteolytic activity identified in the membranes. This activity could not be assigned to previously known brush border proteases. The inactivation and the proteolytic degradation of the kinase could be reproduced also with the pure catalytic subunit of cAMP-dependent protein kinase (C) from rabbit skeletal muscle (M.W. 40000) which was cleaved by the membranal proteolytic activity with concomitant quantitative appearance of a degradation product (M.W. 30000) devoid of kinase activity. The membranal proteolytic activity appears to be specific for C since: (1) it does not degrade the other endogenous proteins in the membrane preparation; (2) it does not degrade any of six arbitrarily chosen proteins from other sources; (3) it catalyzes a limited proteolysis of C which could not be simulated by other proteolytic enzymes such as trypsin, clostripain, chymotrypsin and papain. The attack of C by the membranal protease is blocked by the presence of the nucleotide substrate of the kinase (MgATP). In addition, the undissociated and inactive form of the enzyme (R₂C₂) does not lose its potential enzymatic activity, and neither its catalytic nor its regulatory subunits are digested by the protease. The specific, restricted and limited action of the protease, together with the prevention of its action by the substrate and the regulatory protein (R) of the kinase raise the possibility that the membranal protease may have a distinct physiological (possibly regulatory) assignment.     

Proteinase inhibitors from Vigna unguiculata subsp. cylindrica: I. Occurrence of thiol proteinase inhibitors in plants and purification from Vigna unguiculata subsp. cylindrica

Inhibitors of the thiol proteinase, papain (EC 3.4.22.2), were shown to be present in 11 species of 10 genera of plants. The inhibitor activity was nondialyzable, and precipitated by ammonium sulfate. Tissue cultures from a number of plant genera consisting of rapidly dividing cells contained latent papain inhibitor that could be activated upon heating. Four isoinhibitors of plant thiol proteinases from seeds of the legume Vigna unguiculata subsp. cyclindrica were purified to apparent homogeneity by acrylamide gel electrophoresis with or without sodium dodecyl sulfate. The inhibitors were present in very small amounts compared to the trypsin inhibitors and the degree of purification of the homogeneous isoinhibitors on the assumption that all were present initially in equal amounts was 15,000- to 60,000-fold. The isoinhibitors did not inhibit pepsin, bromelain, and the serine proteinases, trypsin, chymotrypsin, and subtilisin. They were specific for papain, chymopapain, and ficin but their inhibition of the proteinase, esterase, and amidase activities of the three enzymes differed.     

Partial purification of the sugar carrier of intestinal brush border membranes. Enrichment of the phlorizin-binding component by selective extractions

Summary The [³H] phlorizin-binding component of brush border vesicles was enrichedin situ by negative purification. Several procedures, known to effect selective solubilization of membrane components, were used separately or in combination to remove proteins unrelated to the binding. Deoxycholate ruptured the vesicles and released 67% of their protein, thereby increasing the specific [³H] phlorizin-binding activity of the pellet three-to fourfold. Extracting the deoxycholate-pellets with either NaI or alkaline solutions released up to 38% of the deoxycholate-insoluble protein without significantly affecting phlorizin binding. The polypeptide composition of the membranes at the different stages was analyzed by NaDodSO₄-polyacrylamide gel electrophoresis. A number of polypeptides present in the original vesicles could be ruled out as essential components of the [³H] phlorizin binding entity.Intact and deoxycholate-treated vesicles were subjected to proteolytic attack. Papain liberated sucrase and isomaltase from intact vesicles, but affected neither other Coomassiestained bands nor phlorizin binding. Neither the protein composition nor the binding properties of sealed vesicles were influenced by trypsin or chymotrypsin. However, all the proteolytic enzymes tested on deoxycholate-treated membranes substantially reduced [³H] phlorizin binding and produced concomitantly the disappearance of several bands from the electrophoretic profile.Pretreatment of vesicles with papain, followed by deoxycholate extraction and incubation in alkaline media, increased the specific binding activity of the membranes up to ninefold by removing close to 90% of the protein. A limited number of polypeptides are suggested as possible candidates for the glycoside-binding site of intestinal brush borders.     

The possible role of pancreatic proteases in the turnover of intestinal brush border proteins.

1. Intestinal brush border enzymes have heterogeneous rates of turnover, the largest proteins having the fastest turnover. Since the membrane faces the intestinal lumen, the effects of pancreatic factors were examined in mediating this turnover. Surgical subtotal pancreatectomy was used as an experimental model to study the turnover of brush border proteins in the absence of most pancreatic secretions. 2. Subtotal (95%) pancreatectomy of rats was found to cause elevations by about 50% of total activity and specific activities of certain brush border enzymes (maltase, sucrase, lactase), but not of others (alkaline phosphatase, trehalase). Rats were judged to be functionally deficient in pancreatic proteolytic enzymes (a) by demonstration of vitamin B-12 malabsorption, which was corrected by trypsin, and (b) by the finding of only about 20% of proteolytic activity appearing in the lumen after a test meal when compared to control. 3. To measure protein turnover in vivo the method of double labelling was used, where [3H]- and [14C]valine were administered intraduodenally in sequence 10 h apart. With this technique, a high 3H/14C ratio is correlated with rapid turnover. Proteins with apparent molecular weights of about 200 000-270 000 were found to turn over more rapidly than smaller proteins. 3H/14C ranged from 4.7 to 6.2 in animals without pancreatic insufficiency. In the face of decreased pancreatic proteolysis, the 3H/14C ratio was 2.3-3.1, similar to that of proteins with a slow half life. 4. Estimates of relative synthetic rates of large brush border proteins were lower than normal in pancreatectomized animals, but were constant over the period of the labelling experiment. The high enzyme levels in the face of lower synthetic rates confirms that, at the new steady rate, degradation rates must be slower for large brush border proteins in pancreatic insufficiency. 5. In vitro, using purified brush borders, unfractionated pancreatic enzymes were found to remove sucrase, maltase and lactase, but not alkaline phosphatase and trehalase. The enzyme most potent in this respect was the pancreatic protease, elastase. Non-proteolytic enzymes (amylase, lipase, phospholipase A) were inactive in removing enzyme from the brush border. The addition of elastase to pancreatectomized animals in vivo restored the rapid turnover rate of large brush border proteins. 6. A model is thus proposed for the normal catabolism of some large intestinal brush border proteins. It is suggested that the surface of intestinal absorptive cells is being constantly remodelled, and that certain surface enzymes are in part removed from the membrane by the action of pancreatic proteases. A possible special role for elastase is suggested.     

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

A note on stability of purified enzymes to dehydration

Highly purified enzymes from commercial sources were dried from aqueous solutions over CaCl₂ or CaSO₄ at 25° c, 4° c or −4° C. Several heat stable enzymes containing SS groups (chymotrypsin, lipase, pepsin and trypsin) retained most of their activity following drying while papain and ribonuclease retained about one-fourth of their original activity. Most SH containing enzymes lost most if not all activity during drying (catalase, hexokinase, glucose and xanthine oxidases, alcohol, glutamate, isocitrate and glucose-6-phosphate dehydrogenases). Lactic dehydrogenase was the exception in this group retaining 75% of its original activity after drying. Neither sucrose nor mannitol were effective in protecting ribonuclease against inactivation during drying. Temperature during drying had little effect on inactivation.     

Studies on the characterization of the Rho(D) antigen

The Rh_o(D) antigen of red cell membranes was solubilized using ethylenediamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, a pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rh_o(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rh_o(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rh_o(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rh_o(D) molecule in its natural environment. The solubilized Rh_o(D) molecules are apparently not dependent on lipid for their Rh_o(D) activity.     

Proteolysis and lectin histochemistry

Summary Lectin binding to formalin-fixed, paraffin-embedded tissue can often be enhanced by pre-treatment of the sections with proteolytic enzymes. However, the pattern of staining may be profoundly influenced by the type of enzyme preparation which is used.Sites of binding of thirteen different lectins to murine ovary and thyroid gland were studied after exposure of tissue sections to crude trypsin, purified trypsin, purified -chymotrypsin, pepsin, protease VII, papain, bromelain, thermolysin or elastase. With most lectins, the results obtained were similar regardless of which enzyme was used for proteolytic digestion. However, the pattern of binding of soy bean lectin to the ovary and of concanavalin A and common pea lectin to the thyroid gland was highly dependent upon the enzyme used to pre-treat the sections. In both tissues, the staining pattern seen in untreated frozen sections was similar to that found in formalin-fixed, paraffinembedded material digested with purified trypsin, but was different from that observed after exposure of processed sections to crude trypsin. The location of binding sites after treatment of paraffin sections with chymotrypsin was the same as that after digestion with crude trypsin. Results obtained after the use of other proteolytic enzymes varied according to the tissue being studied.These findings imply that the effect of treatment with crude trypsin is due to contaminating chymotrypsin, and demonstrate that the use of purified trypsin may have advantages over other proteolytic enzymes in lectin histochemistry. The observations may also apply to other related cytochemical techniques such as immunocytochemistry.     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.