Showing papers on "Proteolytic enzymes published in 1978"

A study of proteases and protease-inhibitor complexes in biological fluids.

Abstract: We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to alpha2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to alpha2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 mul of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely.

Molecular mechanism of physiological fibrinolysis

Abstract: THE proteolytic enzyme system in blood that is predominantly responsible for removal of fibrin deposits, is called the fibrinolytic system. This system consists of three main components: the proenzyme plasminogen, which can be activated by limited proteolysis to the proteolytic enzyme plasmin; plasminogen activators, the most important of which probably originates in the endothelial cells; and inhibitors, which can rapidly neutralise plasmin or interfere with the activation of plasminogen. The proteolytic enzyme plasmin has a broad specificity, which is not very different from that of trypsin. However, in vivo the main target of plasmin is fibrin. Three hypotheses have been put forward to explain this specificity. Alkjaersig et al.1 have suggested that plasminogen is adsorbed to polymerising fibrin and converted to active enzyme by activators which diffuse into the thrombus. Plasmin would then exert its action in an environment relatively free of inhibitors. Ambrus and Markus2 have proposed that plasmin–inhibitor complexes formed in the circulation dissociate in the presence of fibrin, because plasmin has a greater affinity for fibrin than for its inhibitors. Chesterman et al.3 suggested that the activators bind selectively to fibrin and transform plasminogen, which diffuses into the thrombus, to plasmin. During the past few years specific interactions at the molecular level have been demonstrated between the different components of the fibrinolytic system. These findings now enable us to formulate a molecular model for the regulation of fibrinolysis in vivo.

Observations on the elimination of polyneuronal innervation in developing mammalian skeletal muscle.

Abstract: 1. The mechanism responsible for the elimination of polyneuronal innervation in developing rat soleus muscles was studied electrophysiologically and histologically. 2. Initially all the axons contacting a single end-plate have simple bulbous terminals. As elimination proceeds one axon develops terminal branches while the other terminals remain bulbous and may be seen in contact with, or a short distance away from, the end-plate. It is suggested that the branched terminal remains in contact with the muscle fibre while the other terminals withdraw. 3. At a time when polyneuronal innervation can no longer be detected electrophysiologically, the histological technique still shows the presence of end-plates contacted by more than one nerve terminal. 4. The effect of activity on the disappearance of polyneuronal innervation was examined. Activity was increased by electrical stimulation of the right sciatic nerve. This procedure also produced reflex activity in the contralateral limb. In both cases polyneuronal innervation was eliminated more rapidly in the active muscles. 5. The finding that proteolytic enzymes are released from muscles treated with acetylcholine (ACh), and the observation of the more rapid elimination of supernumerary terminals at the end-plates of active muscles, lead to the suggestion that superfluous nerve-muscle contacts are removed by the proteolytic enzymes in response to neuromuscular activity. The selective stabilization of only one of the terminals is discussed in the light of these results.

A comparison of the killer character in different yeasts and its classification.

Abstract: The interactions between 20 killer yeasts of various genera and species were examined. Ten distinct groups were recognised with respect to killer activity and 10 distinct groups with respect to resistance to killer action. Using both killing and resistance phenotypes, 13 classes of killer yeast were found. With the exception of Torulopsis glabrata NCYC 388, non-Saccharomyces strains of yeast were not killed by a member of the genus Saccharomyces. The killer character of the 3 killing groups of Saccharomyces identified could be cured by treatment with cycloheximide or incubation at elevated temperature and the effectiveness of these procedures was indicative of the category of killer yeast examined. Killer yeasts not belonging to the genus Saccharomyces could not be cured of their activity. Double-stranded ribonucleic acids were extracted only from Saccharomyces spp. and the molecular weights of the species present were a function of the killer class to which a strain belonged. By an analysis of the effects of proteolytic enzymes, temperature and pH on killer activity and by gel chromatography of crude preparations of killer factors, the toxins of different killer classes were shown to be biochemically distinct. However all toxins had certain properties in common consistent with there being a protein component essential to killer action.

Calcium buffering in presynaptic nerve terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism.

Abstract: A latent ATP-dependent Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. The data indicate that a fraction of the Ca uptake (measured with 45Ca) is associated with the intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondrial) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. The proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the ATP-dependent Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca2+ concentration (buffered with EGTA) is less than 0.3 micrometer; brain mitochondria take up little Ca when the ionized Ca2+ level is this low. Preliminary subfractionation studies indicate that the nonmitochondrial Ca storage system does not sediment with synaptic vesicles. We propose that this Ca storage system, which has many properties comparable to those of skeletal muscle sarcoplasmic reticulum, may be associated with intraterminal smooth endoplasmic reticulum. This Ca-sequestering organelle may help to buffer intracellular Ca.

Non-antigenic collagen and articles of manufacture

Abstract: Collagen, available from domestic animals, is freed of noncollagen proteins, glycosaminoglycans and lipids by enzymatic treatment with a proteolytic enzyme to yield a product which is soluble in dilute acidic aqueous solutions (collagen in solution--CIS). The naturally occurring collagen is modified by removal of certain terminal peptide chains, which are described as telopeptides. The modified collagen, so derived, is described as atelopeptide collagen. Native collagen is immunogenic, while atelopeptide collagen is nonimmunogenic or possessed of a negligibly low level of immunogenicity. The collagen in solution is then treated according to a specific regimen under conditions whereby the collagen slowly separates from solution while exposed to mild shear forces. This procedure results in the formation of a fibrous precipitate composed of regularly ordered fibers of collagen possessed of a ropelike structure. These resulting aggregates are referred to as native fibrous micropolymers (NFM). Once the regimen or procedure is completed, and the fiber mass has been formed, the fibrous micropolymers may be freed of salt, taken up in a different solution or modified. For example, cross-links may then be introduced to stabilize the fibers. The products find wide use as packing, membranes, fibers, bags, supports, integuments, and are especially suitable for biologic implantation or application.

Studies of a calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization.

Abstract: A calcium-activated neutral protease was purified 2,700-fold over the crude extract from chicken skeletal muscle. The purified protease migrated as a single band on polyacrylamide gel electrophoresis with or without SDS. Its molecular weight was 80,000 and pH optimum for activity was 7.7. The activity required strictly the presence of calcium (optimum concentration: 1.8 mM) or strontium (optimum concentration: 10 mM) ions. The protease was inhibited by leupeptin, which is known to be a strong inhibitor of papain, cathepsin B, trypsin, and plasmin.

Conjugation of poly-L-lysine to albumin and horseradish peroxidase: a novel method of enhancing the cellular uptake of proteins

Abstract: The carbodiimide-catalyzed conjugation of a 6700 molecular weight fragment of poly-L-lysine to radiolabeled human serum albumin or to horseradish peroxidase enhances the membrane transport of each protein into cultured mouse fibroblasts approximately 11- and 200-fold, respectively. At least 50% of the peroxidase activity remained after conjugation. Trypsinization and carbamylation of the two conjugates demonstrates that the enhancement of their cellular uptake is related to their poly-L-lysine content. Simple addition to the medium of comparable amounts of free poly-L-lysine has no effect on the transport of either native protein. Addition of poly-L-ornithine (molecular weight 200,000) at 3-30 microgram/ml, a condition known to cause enhancement of 125I-labeled human serum albumin uptake by mouse sarcoma cells, has no visible effect on the cellular uptake of native horseradish peroxidase. The intracellular localization of the enzyme-poly-L-lysine conjugate can be demonstrated cytochemically by either light or transmission electron microscopy. A concentration of conjugate that increases the uptake more than 200-fold does not cause any detectable morphological change suggestive of cell toxicity. Furthermore, because poly-L-lysine is an excellent substrate for intracellular proteolytic enzymes, it can be expected to be broken down and reutilized in the cell.

Studies on gonococcus infection. XIII. Occurrence of color/opacity colonial variants in clinical cultures.

Abstract: Colonial variants of Neisseria gonorrhoeae differing in color and opacity characteristics are distributed differently between male urethral and female cervical cultures. Colonial characteristics of cultures isolated from female cervices differ with time of isolation within the menstrual cycle and use of oral contraceptives. These differences may reflect selective forces in the ecology of the cervix, particularly proteolytic enzymes in cervical mucus and menstrual blood. Cycle changes in the characteristics of gonococci isolated from females may have implications for pathogenesis of gonococcal infections and immune response to the gonococcus.

Effect of proteolytic enzymes on the binding of cobalamin to R protein and intrinsic factor. In vitro evidence that a failure to partially degrade R protein is responsible for cobalamin malabsorption in pancreatic insufficiency.

Abstract: Cobalamin (Cbl; vitamin B(12)) malabsorption in pancreatic insufficiency can be partially corrected by bicarbonate and completely corrected by pancreatic proteases but the mechanisms involved are unknown. Because saliva contains enough R-type Cbl-binding protein (R protein) to bind all of the dietary and biliary Cbl, it is possible that R protein acts as an inhibitor of Cbl absorption and that pancreatic proteases are required to alter R protein and prevent such inhibition. To test this hypothesis we studied the ability of R protein and intrinsic factor (IF) to compete for Cbl binding and ability of pancreatic proteases to alter this competition. Human salivary R protein bound Cbl with affinities that were 50- and 3-fold higher than those of human IF at pH 2 and 8, respectively. Cbl bound to IF was transferred to an equal amount of R protein with t((1/2))'s of 2 and 90 min at pH 2 and 8, respectively, and within several hours respective ratios of R protein-Cbl/IF-Cbl of 50 and 2 were observed. Cbl bound to R protein was not transferred to IF at either pH 2 or 8. Incubation of R protein with pancreatic proteases at pH 8 led to a 150-fold decrease in its affinity for Cbl. Incubation of R protein-Cbl with pancreatic proteases led to complete transfer of Cbl to IF within 10 min. Gel filtration studies with R protein-[(57)Co]Cbl and (125)I-R protein showed that pancreatic proteases partially degraded R protein. Pancreatic proteases differed in their ability to effect these changes with trypsin > chymotrypsin > elastase. Pancreatic proteases did not alter IF in any of the parameters mentioned above. Pepsin failed to alter either R protein or IF. THESE STUDIES SUGGEST THE FOLLOWING: (a) that Cbl is bound almost exclusively to R protein in the acid milieu of the stomach, rather than to IF as has been assumed previously; (b) that Cbl remains bound to R protein in the slightly alkaline environment of the intestine until pancreatic proteases partially degrade R protein and enable Cbl to become bound exclusively to IF; and (c) that the primary defect in Cbl absorption in pancreatic insufficiency is a lack of pancreatic proteases and a failure to alter R protein and effect the transfer of Cbl to IF. These studies also suggest that the partial correction of Cbl malabsorption observed with bicarbonate is due to neutralization of gastric HCl, since at slightly alkaline, pH IF can partially compete with R protein for the initial binding and retention of Cbl.

Properties of potato lectin and the nature of its glycoprotein linkages

Abstract: 1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer–dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000–34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the β-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although α-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of β-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single α-galactopyranoside residues that can be removed by the action of α-galactosidase from coffee beans but not by a β-galactosidase. This is the first report of an α-galactoside linkage to serine. The effect of α-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of β-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues had been removed. This inhibitory effect of the arabinofuranoside residues on the β-elimination reaction is likely to be due to a negative charge on the hydroxy groups of the adjacent arabinofuranoside residues, which would be ionized at this high pH value. 6. It is suggested that potato lectin may be representative of a class of soluble plant glycoproteins that would include precursors of the cell-wall glycoprotein extensin. If this is the case, extensin should also contain β-l-arabinofuranosides linked to hydroxyproline and α-d-galactopyranosides linked to serine residues of the polypeptide chain.

Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme.

Abstract: In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.

Demonstration of immunoglobulin in cryostat and paraffin sections of human tonsil by immunofluorescence and immunoperoxidase techniques. Effects of processing on immunohistochemical performance of tissues and on the use of proteolytic enzymes to unmask antigens in sections

Abstract: A fluorescein isothiocyanate (FITC) technique and one based on peroxidase-antiperoxidase (PAP) were used to study the distribution of immunoglobulin (Ig) in cryostat and paraffin sections of human tonsil. Trypsin and other proteolytic enzymes were used to 'unmask' the antigen in paraffin sections. The effects of processing, and particularly of fixation, on the immunohistochemical response of tissues were studied. The FITC and PAP methods detected Ig in paraffin and cryostat sections equally well. The distribution of the antigen was the same with both methods but the PAP method was the more informative. Formaldehyde-sucrose solution proved more suitable for fixing tissues for immunohistochemistry than glutaraldehyde. Trypsin revealed antigen in parraffin sections more efficiently than pepsin, papain, or pronase. Surface Ig (s-Ig) could be demonstrated in trypsinised paraffin sections but less effectively than in cryostat sections. Trypsinised paraffin sections were, however, more suitable for intracellular Ig (c-Ig) than cryostat sections although the performance of cryostat sections could be improved by prior fixation with a coagulative fixative.

Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein

Abstract: 1. Trypsin digestion of human serum transferrin partially saturated with iron(III)- nitrilotriacetate at pH5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCI3, iron(II) ascorbate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH 8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560-564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined.

Interconvertible Enzyme Cascades in Metabolic Regulation

Abstract: Publisher Summary This chapter focuses on interconvertible enzyme cascades in metabolic regulation. The cascades involved in the cyclic covalent modification of interconvertible enzymes are fundamentally different from the irreversible, unidirectional cascades of proteolytic enzymes such as are involved in blood coagulation and complement fixation. When triggered by appropriate stimuli, the latter cascades respond in an explosive manner to produce an avalanche of product needed to meet an occasional biological emergency. However, having fulfilled this function, the cascades are terminated abruptly in response to an entirely different set of autoregulatory signals that lead to self-destruction of the catalytic process. In effect, these unidirectional cascades are contingency systems that serve as biological switches that can be turned on or off intermittently to meet emergency situations. In contrast, the covalent modification of an interconvertible enzyme is a dynamic process in which the coupling of two opposing cascades leads to continual cyclic activation and inactivation of the enzyme. Such cascades are more properly designed for controlled amplification of primary stimuli, as is needed in the regulation of key enzymes in metabolism.

Antibodies to Proteases and Exotoxin A of Pseudomonas aeruginosa in Patients with Cystic Fibrosis: Demonstration by Radioimmunoassay

Abstract: Sera from 33 patients with cystic fibrosis and two pediatric patients being treated for chronic pulmonary infections not related to cystic fibrosis and six sera or serum pools from uninfected individuals were tested with a microtiter radioimmunoassay for reactivity against exotoxin A and two proteases from Pseudomonas aeruginosa. Exotoxin A was purified from a low-protease strain of P. aeruginosa and shown to have adenosine diphosphate-ribose transferase activity and mouse lethality. Proteases were purified from an isolate of P. aeruginosa from a patient with cystic fibrosis and had proteolytic activity against elastin and collagen in an assay employing dimethylated protein substrates. The antibody responses of the patients detected using 125I-labeled antibody to human immunoglobulin were correlated with clinical evaluations expressed as a composite score based on pulmonary findings, case histories, growth and nutrition, and chest X rays. Values in the radioimmunoassay for patients' sera were compared with those of a control serum pool and expressed as the ratio of counts per minute (cpm) in patient serum to the cpm in the control pool. Inverse correlations were found between these ratios for each of the pseudomonas exoproducts and clinical scores; highest ratios occurred in patients showing the lowest clinical scores. These results confirm that proteases and exotoxin A of P. aeruginosa are produced in cystic fibrosis pulmonary infections due to P. aeruginosa and suggest that they may serve as significant virulence factors in these chronic infectious states.

The amino acid sequence of the tryptic peptides from actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis.

Abstract: The amino acid sequences of the tryptic peptides of the thiol proteinase actinidin from Actinidia chinensis were determined by the manual dansyl--Edman procedure. There are 12 tryptic peptides, which give a polypeptide chain of 220 residues with a mol.wt. of 23500. An alignment of the tryptic peptides was made by using the X-ray-crystallographic data of Baker [(1977) J. Mol. Biol. 115, 263--277] determined at 0.28 nm resolution on crystalline actinidin. Detailed evidence for the amino acid sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50083 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

High proportion of ig-producing cells making autoantibody in normal mice.

Abstract: ‘NATURAL’ serum antibodies are thought to arise as a result of stimulation by environmental antigens. Evidence suggests, however, that many such antibodies in normal healthy individuals are specific for and may be induced by a variety of self components. Many of these autoantibodies are directed against ‘buried’ or ‘enzyme-revealed’ self antigens and include those specific for mouse reproductive organs which crossreact with human group A erythrocytes1, human and rabbit immunoglobulins treated with proteolytic enzymes2,3, denatured DNA4, enzyme-treated human lymphocytes and erythrocytes5–8, and mouse erythrocytes treated with the proteolytic enzyme bromelain9. This widespread occurrence of autoantibodies could suggest that the normal immune system is preoccupied in reacting against self antigens. One way of measuring the extent of this reactivity is to compare the number of B cells making a given autoantibody(s) with the total number of B cells producing immunoglobulin (Ig) irrespective of specificity. We have previously shown that normal conventional and germ-free mice possess in their spleens considerable numbers of PFC against bromelain-treated isologous mouse erythrocytes (BrM), an ‘internal’ antigen of mouse erythrocytes9,10. We have measured the proportion of Ig-secreting cells in the lymphoid organs of normal CBA/H mice forming PFC against BrM and found that in some (but not all) major lymphoid organs between 1% and >50% of the existing or potential Ig-secreting cells are specific for BrM.

Characterization of adenovirus-associated virus-induced polypeptides in KB cells.

Abstract: Electrophoretic analysis of KB cells coinfected with adenovirus-associated virus (AAV) type 2, a defective parvovirus, and adenovirus type 5 (as helper) have revealed the synthesis in vivo of at least five AAV-specific polypeptides. The three largest polypeptides, with molecular weights of 90,700, 71,600, and 60,000 comigrated in polyacrylamide gels with the three AAV structural polypeptides. The remaining two polypeptides had molecular weights of 24,900 and 15,800. The concentrations of the AAV-induced polypeptides relative to one another remained approximately constant during the infectious cycle, and the structural components were present in proportions similar to those found in purified virions. As determined by pulse-chase experiments, all polypeptides were generated at the level of protein synthesis and not by posttranslational proteolytic processing. Although inhibitors of proteolytic enzymes failed to influence the pattern of AAV-induced polypeptides, and amino acid analog, L-canavanine, blocked the appearances of both the major structural polypeptide (60,000 daltons) and the larger nonstructural polypeptide (24,900 daltons). Taken in conjunction with pulse-chase data, this result supports a model whereby the major virion polypeptide is produced by proteolytic cleavage of the nascent polypeptide chain.

Spectrofluorometric Measurement of the Binding of Ethidium to Superhelical DNA from Cell Nuclei

Abstract: Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasi-circular and supercoiled by measuring spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and γ-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their γ-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to γ-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 × 105 base pairs. The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed.

Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

Abstract: We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or α-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 106 cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, α1-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

Proteolytic enzyme activity of blood leukocytes and cerebrospinal fluid in multiple sclerosis

Abstract: Upon stimulation by immune complexes, the polymorphonuclear (PMN) blood secretes lysosomal hydrolases, including neutral proteinase, which is concentrated in the PMN cell. Neutral and acid proteinase activity were increased and decreased, respectively, in the circulating white cells of patients with multiple sclerosis during an exacerbation of the disease, but there was no correlation with serum immune complex levels. Neutral proteolytic activity in the cellular fraction of the cerebrospinal fluid was also found to be elevated in acute multiple sclerosis, as monitored by digestion of myelin basic protein.

The release of prostaglandins and thromboxanes from guinea-pig lung by slow reacting substance of anaphylaxis, and its inhibition.

Abstract: 1 Rabbit aorta contracting substance (RCS; consisting mainly of thromboxane A2) and prostaglandin-like material were released from guinea-pig isolated perfused lungs by injection of slow reacting substance of anaphylaxis (SRS-A). 2 SRS-A was resistant to boiling and proteolytic enzymes and was therefore distinguished from rabbit aorta contracting substance releasing factor (RCS-RF). 3 The release of RCS and prostaglandin-like material by SRS-A was anatagonized by indomethacin (1 microgram/ml), betamethasone and dexamethasone (4 to 50 microgram/ml). 4 Imidazole (200 microgram/ml) inhibited the formation of thromboxane A2 but not that of prostaglandins. 5 The activity of SRS-A on guinea-pig ileum and its ability to release RCS and prostaglandins were destroyed by incubation with arylsulphatase (0.83 microgram to 1 mg/ml) and with lipoxidase (16.5 to 50 microgram/ml): SRS-A lost activity on incubation with bovine serum albumin (9 microgram/ml) due to protein binding.