Showing papers on "Proteolytic enzymes published in 1985"

A 38,000-dalton membrane protein (p38) present in synaptic vesicles.

Abstract: A protein with an apparent molecular mass of 38,000 daltons designated p38 was found in synaptic vesicles from rat brain. The subcellular distribution of p38 and some of its properties were determined with the aid of polyclonal and monoclonal antibodies. The subcellular distribution of p38 was similar to that of synapsin I, a synaptic-vesicle specific phosphoprotein. p38 in the synaptic vesicle fraction purified by controlled-pore glass bead chromatography showed an enrichment of more than 20-fold over the crude homogenate. Immunostaining of sections through various brain regions revealed an intense labeling of most, and possibly all, nerve terminals. Only faint reaction in the region of the Golgi apparatus and no detectable labeling of axons and dendrites was observed. Two-dimensional electrophoresis revealed that p38 has an acidic pI. Solubilization experiments, as well as phase separation experiments using Triton X-114, indicated that p38 is an integral membrane protein. Binding of antibodies to intact synaptic vesicles, as well as controlled proteolytic digestion of intact and detergent-treated vesicles, revealed that p38 has a domain exposed on the cytoplasmic surface.

Techniques in Immunocytochemistry

Abstract: Contributors. Foreword. Preface. Tissue Preparation Methods for Immunocytochemistry, P.Brandtzaeg. Applications of Double-label Immunofluorescence, K.B. Pryzwansky. The Unlabelled Antibody Peroxidase-Anti-Peroxidase Method (PAP), J. Burns. The Protein A-Gold (pAg) Technique-A Qualitative and Quantitative Approach for Antigen Localization on Thin Sections, J. Roth. Double Immunoenzymatic Labelling, D.Y. Mason and R.-E. Woolston. "Double Bridge" Techniques of Immunocytochemistry, L.L. Vacca. Light Microscopic Immunocytochemistry with Fixed, Unembedded Tissues, R. Grzanna. Methods for Locating and Identifying Antigens in Plant Tissues, R.B. Knox. The Use of Proteolytic Enzymes for Improved Localization of Tissue Antigens with Immunocytochemistry, J.C.W. Finley and P. Petrusz. Pre-embedding Staining for Immunocytochemistry at the Light Microscopic Level, R.M. Franklin. Post-embedding Unlabeled Antibody Techniques for Electron Microscopy, P. Ordronneau. Specific Immunocytochemical Localization of Neuropeptides: A Utopian Goal?, F. van Leeuwen. Subject Index.

Redesigning trypsin: alteration of substrate specificity

Abstract: A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.

Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds

Abstract: A survey of antibiotic-producing bacteria from the microbial flora attached to seaweeds and the study of their antibiotic capacities were carried out From 5 species of green and brown marine algae, 224 bacterial strains were isolated and tested for antibiotic production A total of 38 strains displayed antibiotic activity, withEnteromorpha intestinalis being the source of the highest number of producer strains All epiphytic bacteria with antibiotic activity were assigned to thePseudomonas-Alteromonas group Antagonism assays among the isolates demonstrated that each producer strain inhibits the growth of the other producers, as well as of some nonproducer strains also isolated from seaweeds Likewise, an autoinhibitory effect was observed in all antibiotic-producing strains Antibacterial spectra of all the strains include activity againstStaphylococcus, Alcaligenes, Pseudomonas, Vibrio, Pasteurella, andAchromobacter A preliminary characterization of the antibiotic substances produced by these epiphytic bacteria demonstrated that they are low molecular weight compounds, thermolabile, and anionic and are not affected by proteolytic enzymes The role that these inhibitory substances can play in the natural environment is discussed

A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.

Abstract: Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.

Purification and characterization of a multicatalytic high-molecular-mass proteinase from rat skeletal muscle.

Abstract: A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

Abstract: An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid–clotting factor interactions.

Distribution of cathepsins B and H in rat tissues and peripheral blood cells.

Abstract: The concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-alpha + TPI-beta), whereas in tissues containing large amounts of TPI-alpha, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-alpha plus TPI-beta, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue

Abstract: Nitrogenase in Rhodospirillum rubrum is inactivated in vivo by the covalent modification of the Fe protein with a nucleotide. The preparation of two modified peptides derived from proteolytic digestion of the inactive Fe protein is described. The modifying group is shown to be adenosine diphosphoribose, linked through the terminal ribose to a guanidino nitrogen of arginine. The structural features were established by using proton and phosphorus NMR, positive- and negative-ion fast atom bombardment mass spectrometry, and fast atom bombardment/collisionally activated decomposition mass spectrometry. Spectral methods along with chromatographic analysis and sequential degradation established the sequence of the modification site of Fe protein as Gly-Arg(ADR-ribose)-Gly-Val-Ile-Thr. This corresponds to the sequence in the Fe protein from Azotobacter vinelandii for amino acid residues 99 to 104.

Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable.

Abstract: The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein-degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli.

The synthesis of protein with an identification peptide

Abstract: A hybrid polypeptide composed of an identification peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the identification peptide and the desired functional protein. The identification peptide consists of a highly antigenic N-terminal portion and a C-terminal linking portion that connects the identification peptide to the N-terminal of the functional protein. The linking portion of the identification peptide is cleavable at a specific amino acid residue adjacent the functional protein by use of a sequence specific proteolytic enzyme or chemical proteolytic agent. The hybrid polypeptide expressed by the host cells transformed by the cloning vector is removed therefrom and purified by affinity chromatography techniques by use of an immobilized ligand specific to the antigenic portion of the identification peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolic enzyme or chemical agent, thereby releasing the mature functional protein in highly purified, highly active state.

Evidence for Separate Adhesion Mechanisms for Hydrophilic and Hydrophobic Surfaces in Vibrio proteolytica.

Abstract: The proteolytic enzymes pronase, trypsin, and chymotrypsin and the surfactant Triton X-100 inhibited attachment of Vibrio proteolytica to the hydrophobic substratum polystyrene by >97%. These treatments had no effect on attachment to hydrophilic substrata such as glass or tissue culture dishes. Both pronase and Triton X-100 effected the removal of previously attached cells from polystyrene but not from hydrophilic surfaces. Removal of cells from polystyrene by pronase left material (which we have termed footprints) that stained with the protein-specific stain Hoechst 2495 but not with the DNA-specific stain Hoechst 33342. Pronase treatment also caused a significant decrease in cell surface hydrophobicity as determined by phase partitioning in hexane or petroleum ether. Collectively, these results imply the existence of separate mechanisms for the adhesion of V. proteolytica to hydrophilic and hydrophobic substrata and suggest a role for protein in the latter mechanism.

Detection of a Trichosporon beigelii antigen cross-reactive with Cryptococcus neoformans capsular polysaccharide in serum from a patient with disseminated Trichosporon infection.

Abstract: Latex beads coated with anti-Cryptococcus neoformans antibody were agglutinated by serum from a bone marrow transplant recipient having a disseminated infection caused by Trichosporon beigelii. The cryptococcal latex agglutination titer in the serum of the patient rose to 1:2,560 by the time of his death. Necropsy confirmed the disseminated Trichosporon infection and absence of C. neoformans. Cell wall extracts of the isolate of the patient and two additional strains of T. beigelii agglutinated anti-Cryptococcus-coated latex beads. The antigen in the serum of the patient and in the extracts responsible for the agglutination was not destroyed by proteolytic enzymes or heat. A single antigen reactive with rabbit anti-Trichosporon serum could be identified in the serum of the patient and the cell wall extracts by rocket immunoelectrophoresis and crossed immunoelectrophoresis. Rocket immunoelectrophoresis and indirect fluorescent-antibody staining demonstrated that anti-Trichosporon antibody recognized the capsular polysaccharide of C. neoformans. Images

Neuron-specific interactions with two neurite-promoting fragments of fibronectin

Abstract: Proteolytic fragments of fibronectin were used to identify regions of the molecule that support neurite extension and to investigate further the differential behavior of central and peripheral nervous system neurons in response to fibronectin (Rogers, S. L., P. C. Letourneau, S. L. Palm, J. B. McCarthy, and L. T. Furcht (1983) Dev. Biol. 98: 212–2220). Fibronectin fragments with differing biological activities were produced by proteolytic digestion with trypsin and cathepsin D and sequential affinity chromatography on gelatin-agarose and heparin- Sepharose. The resulting fragments (described by Smith, D. E., D. F. Mosher, R. B. Johnson, and L. T. Furcht (1982) J. Biol. Chem. 257: 5831– 5838; Smith, D. E., and L. T. Furcht (1982) J. Biol. Chem. 257: 6518– 6523 included an NH2-terminal 27,000-dalton peptide that weakly binds heparin, a 46,000-dalton gelatin-binding fragment, a series of fragments (80,000 to 125,000 daltons) from the center of the molecule containing previously described cell-binding activity, two major peptides of Mr = 33,000 and 66,000 that bind heparin strongly and are thought to originate from the A and B chains, respectively, of plasma fibronectin, and a 31,000-dalton COOH-terminal peptide containing a free sulfhydryl from the A chain of the molecule. Tissue culture dishes were treated with these proteolytic preparations, and dissociated embryonic chick peripheral (PNS) and central nervous system (CNS) cells were cultured on each experimental substratum in serum-free medium. The fibronectin fragments were evaluated for ability to promote cell attachment, neurite initiation, and maintenance of neurite growth. The 27,000–, 46,000-, and 31,000-dalton preparations did not promote cell attachment or neurite extension. Both PNS and CNS neurons attached to and extended stable neurites upon the COOH-terminal heparin-binding preparation containing the 33,000- and 66,000-dalton peptides. A differential response of the neurons to the 80,000- to 125,000-dalton “cell-binding” peptides was observed: whereas PNS neurons maintained neuritic growth on this preparation for at least 48 hr, CNS neurons extended neurites during the first 24 hr of culture but, by 48 hr, withdrew these neurites and became increasingly clumped. On the basis of (1) the observed neuronal responses to the heparin binding and “cell binding” regions, and (2) the different ligand-binding properties of these regions, we propose that cell attachment and neurite extension can be mediated and/or modulated by two separate regions of fibronectin and that cellular response to the intact molecule may involve multivalent interactions.

Rectification of single GABA-gated chloride channels in adult hippocampal neurons.

Abstract: The properties of single chloride channels activated by gamma-aminobutyric acid (GABA) were investigated with hippocampal slices from adult guinea pigs. After the slices were treated with proteolytic enzymes, gigaseal recordings were made from excised patches of pyramidal or granule cell membranes. This newly developed preparation permits the application of patch-clamp techniques to the adult mammalian central nervous system. Guinea pig hippocampal slices were prepared in a conventional manner. Once prepared, the slices were treated with two different enzymes for brief periods and gently agitated. Slices generally split apart along the boundaries of the cell body regions, exposing neuronal somata. Standard patch-clamp techniques were used for the gigaseal recordings from excised patches. Solutions for both sides of the patches consisted of symmetrical concentrations of chloride, with all cation channels blocked. GABA at concentrations of 0.5-1.0 microM was added to the solution for the extracellular side of the patches. At transmembrane potentials negative to the chloride reversal potential (0 mV), the conductance through the GABA-gated chloride channels was approximately 20 pS. When the transmembrane potential was changed to positive values, the chloride conductance increased dramatically. For example, at +40 mV the conductance through the GABA-gated channels was almost 40 pS. Ramp-clamp commands were used to measure the current-voltage (I-V) relationship through single open channels. The open-channel I-V curves displayed outward rectification. The relationship between open-channel conductance and voltage could be fitted reasonably well by a single energy-barrier model for the channel, with the higher energy barrier placed near the cytoplasmic side of the membrane (at a fractional distance through the membrane of 0.73).(ABSTRACT TRUNCATED AT 250 WORDS)

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants.

Abstract: Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus-transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.

Changes in hemopoietic tissue of rainbow trout under influence of stress

Abstract: As in higher vertebrates, stress increases the susceptibility of fishes to disease. This effect is accompanied by changes in circulating white blood cells: the number of lymphocytes decreases while the number of granulocytes increases. Investigations were therefore undertaken to determine in what ways stress induces changes in hemopoietic organs, particularly spleen and head kidneys of rainbow trout Salmo gairdneri Handling and social conflict were used as stressors. These psychic stimuli are typically encountered in aquaculture and under laboratory conditions. Under the influence of both stressors hemopoiesis in spleen and head kidney is disturbed. Blast cells become rare or disappear completely from the tissue. The numbers of mature, intact PMN cells and of small lyrnphocytes also decrease. Hypertrophic macrophage-like cells with vacuolized cytoplasm appear in increasing numbers. They develop mainly from immature PMN and reticuloendothelial cells. They swell continuously while undergoing evident disintegration. At the same time, destruction of erythrocytes proceeds at an increased rate. The high numbers of macrophage-like cells may indicate that the stresson initially stimulate the defense mechanisms and thereby serve an adaptive function. Possibly, under continued stress, their proteolytic enzymes begin to damage the body's own blood cells. The decreased production of new blood cells and the increased destruction of immune-competent cells still present seem finally to contribute to disease susceptibility under stress.

High surface hydrophobicity of autoaggregating Staphylococcus aureus strains isolated from human infections studied with the salt aggregation test.

Abstract: A total of 209 strains of Staphylococcus aureus isolated from infections and 23 strains from nose cultures of healthy laboratory personnel were compared for relative surface hydrophobicity in the salt aggregation test (Lindahl et al., Biochim. Biophys. Acta 677:471-476, 1981). In the standard method, bacterial cell suspensions from blood agar-grown cultures were tested for visible aggregation by "salting out" in serial dilutions of ammonium sulfate (0.1 to 1.6 M [final concentration]). Bacteria were defined as extremely hydrophobic when showing autoaggregation in saline or in 0.002 M sodium phosphate buffer (pH 6.8). Using this definition, we found a large number of strains isolated from various infections to be very hydrophobic: 123 of 135 strains from patients with septicemia (91%), 54 of 60 strains from wound infections (90%), and 12 of 14 strains from urinary tract infections (86%). In contrast, only 9 of 23 strains from nose cultures of healthy carriers (39%) were autoaggregating. A total of 12 autoaggregating strains were grown on various solid and liquid media. Only growth on hematin agar was found to completely suppress surface hydrophobicity as revealed by our salt aggregation test method, and growth in liquid media prevented the expression of hydrophobicity in most strains. Growth at 20 or 42 degrees C or under anaerobic conditions did not affect hydrophobicity. Cells harvested from various phases of growth did not differ significantly in surface hydrophobicity. Heating washed cell suspensions at 56 degrees C did not affect the salt aggregation test values, whereas heating the cell suspensions at 80 and 100 degrees C caused a significant decline in hydrophobicity. The addition of ethylene glycol (25% [vol/vol] final concentration) prevented the autoaggregation of 10 of the 12 strains. Likewise, treating the cell suspensions with proteolytic enzymes decreased the surface hydrophobicity, indicating that surface proteins contribute to high surface hydrophobicity of autoaggregating strains.

IgG proteolytic activity of Pseudomonas aeruginosa in cystic fibrosis

Abstract: To study how fragmented IgG antibodies might arise within the respiratory secretions of individuals with cystic fibrosis (CF), we screened protease extracts from CF polymorphonuclear leukocytes and mucoid and nonmucoid transformants of Pseudomonas aeruginosa from patients with CF for IgG proteolytic activity. All strains of P. aeruginosa tested exhibited IgG proteolytic activity. Incubation for 7 hr at 37 C was required to demonstrate generation of free Fc gamma immunoreactivity. Further analysis of these cleavage products of CF IgG demonstrated generation of Fc gamma polypeptides with 4S sedimentation coefficients and F(ab')2 fragments with 5S coefficients. Bacterial IgG proteolytic activity was inhibited by EDTA and was associated with levels of bacterial elastase exceeding 5 micrograms/mg of total protein. Pseudomonas elastase was significantly more active on IgG1 and IgG3; IgG2 and IgG4 were more resistant. This bacterial exoproduct appears to digest IgG molecules into Fab gamma, F(ab')2 fragments, and a free Fc gamma piece with a molecular weight of 40,000.

Adherent and soluble mucus in the stomach and duodenum.

Abstract: Gastroduodenal mucus is present as a water insoluble gel adherent to the mucosal surface and as a viscous mobile solution in the lumen. The protective properties of the mucus against acid (with bicarbonate), pepsin (diffusion barrier) and mechanical damage depend on the quality (structure) and quantity (thickness) of the adherent mucus gel layer. Adherent mucus is a viscoelastic gel which is 95% (v/v) water. It is permeable to ions and smaller molecules (Mr c. 1000), but is impermeable to large proteins (Mr,c. 17,000) including pepsins. However, mucus is solubilized rapidly by pepsin, more slowly (>-1 h) by thiol agents, and is unchanged following exposure to bile, acid and ethanol (<40%). Glycoprotein macromolecules (Mr≥2×106) are the structural components of the mucus gel and have a polymeric, structure of glycoprotein subunits (Mrc. 5×105, for gastric mucus) joined by disulphide bridges between their protein cores. This glycoprotein polymerization, which is essential for gel formation and hence function, is the site of action of proteolytic enzymes and thiol agents. The glycoprotein polymeric structure is deficient in antral mucus from patients with peptic ulcer disease.In vivo, adherent mucus forms a thin but continuous cover of variable thickness (50–450 μm in man, about two-fold less in rat) over the gastroduodenal mucosa. Pepsin in gastric juice will rapidly dissolve this mucus cover and can be active up to luminal pH values of 5. Mucus erosion by pepsin or by abrasion must be balanced by its secretion. Prostaglandins and carbachol stimulate a rapid increase (within minutes) in mucus thickness of up to two-fold. Soluble luminal mucus can be increased by mucus secretagogues, mucosal damaging agents, or peptic degradation of adherent mucus. Increases in luminal mucus can occur independently of increased gel thickness.

The biology of mycorrhiza in the ericaceae: x. the utilization of proteins and the production of proteolytic enzymes by the mycorrhizal endophyte and by mycorrhizal plants.

Abstract: The ability of the ericoid mycorrhizal endophyte to utilize a range of proteins as substrates for growth is assessed in liquid culture and in mycorrhizal association with host plants Some aspects of proteolytic enzyme production are also investigated The fungus readily utilizes the soluble protein bovine serum albumin (BSA) as sole nitrogen and carbon source, and produces lower yields on less soluble plant and animal proteins Maximum yields of endophyte on all substrates were obtained in the pH range 3 to 5 Infection provides a significant enhancement of plant growth on agar over this pH range on most of the proteins Yields and nitrogen contents of mycorrhizal plants grown on cellulose sheets with BSA as sole N source were significantly higher than those of the uninfected controls, which were unable to use protein Using a chromogenic substrate it was shown that the pH optimum for enzyme activity is comparable with that for utilization of protein in pure culture and in mycorrhizal association Non-mycorrhizal plants produced negligible proteolytic activity The significance of these observations is discussed in relation to the nutrition of both host and fungus in the natural environment, and the broader ecological implications of the results are assessed

Interaction of opiates with opioid binding sites in the bovine adrenal medulla: I. Interaction with delta and mu sites.

Abstract: In the present study we examined the interaction of opiates with the delta and mu opioid binding sites in the bovine adrenal medulla. [3H][D-Ala2, D-Leu5]-enkephalin ( [3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze delta site interactions, whereas either [3H]DADLE in the presence of saturation concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]-enkephalin ( [3H]DAGO) was used for the determination of mu sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, alpha-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second-order bimolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the delta and mu selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.

The predominant protein in human seminal coagulate

Abstract: Lilja H, Laurell C-B. The predominant protein in human seminal coagulate. Scand J Clin Lab Invest 1985; 45: 635–641.The predominant protein in human seminal vesicle secretion constitutes the structural protein of coagulated semen. This high molecular weight protein (HMW-SV-protein) is stable in seminal vesicle secretion during in vitro storage at 37 d`C for at least 20 h, but is rapidly cleaved on mixing with prostatic proteases. Seminal coagulate, washed free of souble components, is dissoluble by 2 to 3 mol/1 of guanidine-HCl. Although dithiothreitol added to seminal coagulate does not liquify the clot, complexes between HMW-SV-proteins are broken up by reduction under denaturing conditions, which suggests that the non-covalent linkages of HMW-SV-proteins are essential in the clot. Prostatic proteases cleave the HMW-SV-protein during liquefaction of ejaculated semen to a series of labile proteins. These proteins are further cleaved to peptides of successively decreasing size after completed liquefaction...

Characterization of two highly diverged but developmentally co-regulated cysteine proteinase genes in Dictyostelium discoideum.

Abstract: The cysteine proteinase 1 and 2 mRNA sequences of Dictyostelium discoideum encode proteins with a high degree of homology to plant and animal sulphydryl proteinases. The two mRNA sequences are co-ordinate in their regulation, both being first expressed late during cellular aggregation, prematurely induced in response to exogenous cAMP and several-fold enriched in prestalk over prespore cells. The two proteins are considerably diverged, with only 43% overall homology but all residues known to be important in catalysis are conserved and both contain a hydrophobic leader peptide which forms part of an N-terminal domain of just over 100 amino acids not found in the mature form of known cysteine proteinases. We have determined the sequence organization of both genes and find differences both in the number and position of introns. The close co-regulation of these two genes suggests that they may play a common role in Dictyostelium development, presumably in the autodigestion of cellular protein which occurs during differentiation. However, the low degree of sequence homology and major differences in gene organization indicate that they have undergone a considerable period of separate evolution and that they may differ in their precise function.