alpha-chymotrypsin and Hemolysis

Topics: Animals; Cell Membrane; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Freeze Etching; Hemolysin Proteins; Hemolysis; Humans; Isoelectric Focusing; Lipids; Membranes, Artificial; Microscopy, Electron; Molecular Weight; Phospholipases; Pronase; Rabbits; Staphylococcus; Subtilisins; Toxins, Biological; Trypsin; Ultracentrifugation

α-Helical antimicrobial peptides (αAMPs) are among the potential candidates for new anti-infectives to tackle the global crisis in antibiotic resistance, but they suffer from low bioavailability due to high susceptibility to enzymatic degradation. Here, we describe a strategy to increase the resistance of αAMPs against proteases. Fusing the 12-residue αAMP KR-12 with a Trp-cage domain induces an α-helical structure in the otherwise unfolded KR-12 moiety in solution. The resulting antimicrobial Trp-cage exhibits higher proteolytic resistance due to its stable fold as evidenced by correlating sequence-resolved digest data with structural analyses. In addition, the antimicrobial Trp-cage displays increased activity against bacteria in the presence of physiologically relevant concentrations of NaCl, while the hemolytic activity remains negligible. In contrast to previous strategies, the presented approach is not reliant on artificial amino acids and is therefore applicable to biosynthetic procedures. Our study aims to improve the pharmacokinetics of αAMPs to facilitate their use as therapeutics.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Chymotrypsin; Drug Design; Erythrocytes; Hemolysis; Humans; Liposomes; Microbial Sensitivity Tests; Protein Conformation, alpha-Helical; Protein Stability; Proteolysis; Trypsin

The first amphibian skin secretion-derived Bowman-Birk type chymotrypsin inhibitor is described here from the Asian green frog, Hylarana erythraea, and was identified by use of molecular cloning and tandem mass spectrometric amino acid sequencing. It was named Hylarana erythraea chymotrypsin inhibitor (HECI) and in addition to inhibition of chymotrypsin (Ki = 3.92 ± 0.35 μM), the peptide also inhibited the 20 S proteasome (Ki = 8.55 ± 1.84  μM). Additionally, an analogue of HECI, named K

Topics: Amino Acid Sequence; Animals; Biological Products; Cell Line, Tumor; Cell Proliferation; Chymotrypsin; Cloning, Molecular; Drug Screening Assays, Antitumor; Enzyme Assays; Erythrocytes; Hemolysis; Horses; Humans; Lung Neoplasms; Peptides; Proteasome Endopeptidase Complex; Ranidae; Sequence Analysis, Protein; Skin; Tandem Mass Spectrometry; Toxicity Tests; Trypsin Inhibitors

A 255-bp cDNA encoding an 84-amino acid residue (aa) precursor protein containing 8 half-cysteines was cloned from the skin of the frog, Ceratophrys calcarata. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 63-aa mature peptide with amino acid sequence, NVTPATKPTPSKPGYCRVMDELILCPDPPLSKDLCKNDSDCPGAQKCCYRTCIMQCLPPIFRE. The mature was named ceratoxin. Ceratoxin shares significant sequence similarity with the toxin family of waprins containing the whey acidic protein-type (WAP) four-disulfide core domain found in snake venoms. Antimicrobial and trypsin-inhibitory abilities of recombinant ceratoxin were tested. Recombinant ceratoxin showed strong antimicrobial activities against wide spectrum of microorganisms including Gram-negative and Gram-positive bacteria and fungi. It had no serine protease-inhibitory activity. The current results suggested that the snake venom-like waprin with antimicrobial activities in the frog skin plays a role in innate immunity.

Topics: Amino Acid Sequence; Amphibian Venoms; Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Anura; Base Sequence; Chymotrypsin; Cloning, Molecular; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemolysis; Microbial Sensitivity Tests; Molecular Sequence Data; Rabbits; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Skin; Snake Venoms; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcus aureus; Substrate Specificity; Subtilisin; Toxins, Biological

This work describes how the systematic incorporation of a range of unnatural amino acid derivatives in the P1, P1', and P2' positions allows for the generation of short lactoferricin based cationic antimicrobial peptides with a stability toward chymotryptic degradation. The necessary pharmacophore sets the peptides up for degradation by chymotrypsin, and a heavily truncated native tripeptide was rapidly digested despite its short sequence. Degradation studies indicated that increased half-lives could be obtained by altering the binding to each subsite surrounding the active site without sacrificing the antimicrobial activity. Important structural and mechanistic features were revealed in a fashion not feasible through the use of native peptide substrates. The results, which are generally applicable to a range of relevant peptides, further show that not only the S1 pocket, but also to the notably less studied S1' site can be used to control the proteolytic stability by incorporating different analogues of tryptophan and arginine.

Topics: Amino Acids; Antimicrobial Cationic Peptides; Calorimetry; Chymotrypsin; Drug Stability; Escherichia coli; Hemolysis; Humans; Hydrophobic and Hydrophilic Interactions; Microbial Sensitivity Tests; Models, Molecular; Oligopeptides; Protein Binding; Staphylococcus aureus; Structure-Activity Relationship; Thermodynamics

A 5.6 kDa trypsin-chymotrypsin protease inhibitor was isolated from the tubers of the potato (Solanum tuberosum L cv. Gogu) by extraction of the water-soluble fraction, dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This inhibitor, which we named potamin-1 (PT-1), was thermostable and possessed antimicrobial activity but lacked hemolytic activity. PT-1 strongly inhibited pathogenic microbial strains, including Candida albicans, Rhizoctonia solani, and Clavibacter michiganense subsp. michiganinse. Automated Edman degradation showed that the N-terminal sequence of PT-1 was NH2-DICTCCAGTKGCNTTSANGAFICEGQSDPKKPKACPLNCDPHIAYA-. The sequence had 62% homology with a serine protease inhibitor belonging to the Kunitz family, and the peptide inhibited chymotrypsin, trypsin, and papain. This protease inhibitor, PT-1, was composed of polypeptide chains joined by disulfide bridge(s). Reduced PT-1 almost completely lost its activity against fungi and proteases indicating that disulfide bridge is essential for its protease inhibitory and antifungal activity. These results suggest that PT-1 is an excellent candidate as a lead compound for the development of novel oral or other anti-infective agents.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Chymotrypsin; Hemolysis; Humans; Micrococcus; Mitosporic Fungi; Molecular Sequence Data; Molecular Weight; Plant Proteins; Sequence Alignment; Serine Proteinase Inhibitors; Solanum tuberosum; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin

The present work aimed to study the effects of cultivation conditions on the haemolytic activities of Pseudoalteromonas issachenkonii.. The kinetics of growth and haemolytic activities was investigated on sea-salts and NaCl-based nutrient media supplemented with either starch, or KBr over a period of 140 h. The first haemolytic activity occurred when bacterial cells reached the late stationary phase. The second haemolytic activity was observed in marine broth (MB) after 110 h of incubation. Addition of Fe to the culture medium neither affected bacterial growth nor reduced the haemolytic activity. However, the activity was enhanced in the presence of iron chelator. The second haemolytic activity was not affected by Ca2+, or inhibited by chymotrypsin or EDTA.. The production of haemolysins by P. issachenkonii was greater on MB and was dependent on both the medium composition and time of incubation. The second haemolytic activity was heat stable, nonproteinaceous, calcium-independent and was regulated by Fe.. The results demonstrated the importance of optimization of both the media composition and monitoring the haemolytic activity over a prolonged cultivation time to detect different types of haemolysins.

Topics: Animals; Bacterial Proteins; Calcium Chloride; Chymotrypsin; Culture Media; Edetic Acid; Erythrocytes; Hemolysin Proteins; Hemolysis; Humans; Iron; Mice; Pseudoalteromonas; Rabbits; Temperature

Dendrimeric peptides selective for microbial surfaces have been developed to achieve broad antimicrobial activity and low hemolytic activity to human erythrocytes. The dendrimeric core is an asymmetric lysine branching tethered with two to eight copies of a tetrapeptide (R4) or an octapeptide (R8). The R4 tetrapeptide (RLYR) contains a putative microbial surface recognition BHHB motif (B = basic, H = hydrophobic amino acid) found in protegrins and tachyplesins whereas the octapeptide R8 (RLYRKVYG) consists of an R4 and a degenerated R4 repeat. Antimicrobial assays against 10 organisms in high- and low-salt conditions showed that the R4 and R8 monomers as well as their divalent dendrimers contain no to low activity. In contrast, the tetra- and octavalent R4 and R8 dendrimers are broadly active under either conditions, exhibiting relatively similar potency with minimal inhibition concentrations < 1 microm against both bacteria and fungi. Based on their size and charge similarities, the potency and activity spectrum of the tetravalent R4 dendrimer are comparable to protegrins and tachyplesins, a family of potent antimicrobials containing 17-19 residues. Compared with a series of linearly repeating R4 peptides, the R4 dendrimers show comparable antimicrobial potency, but are more aqueous soluble, more stable to proteolysis, less toxic to human cells and more easily synthesized chemically. These results suggest repeating peptides that cluster the charge and hydrophobic residues may represent a primitive form of microbial pattern-recognition. Incorporating such knowledge in a dendrimeric design therefore presents an attractive approach for developing novel peptide antibiotics.

Topics: Anti-Bacterial Agents; Biochemistry; Chymotrypsin; Drug Design; Hemolysis; Humans; Microbial Sensitivity Tests; Peptides; Structure-Activity Relationship; Trypsin

Mammalian reovirus virions undergo partial disassembly of the outer capsid upon exposure to proteases in vitro, producing infectious subvirion particles (ISVPs) that lack protein sigma3 and contain protein mu1/mu1C as endoprotease-generated fragments mu1delta/delta and phi. ISVPs are thought to be required for two early steps in reovirus infection: membrane penetration and activation of the particle-bound viral transcriptase complexes. Genetic and biochemical evidence implicates outer-capsid protein mu1 in both these steps. To determine whether the cleavage of mu1/mu1C is relevant to the unique properties of ISVPs, we analyzed the properties of novel subvirion particles that lacked sigma3 yet retained mu1/mu1C in an uncleaved but cleavable form. These detergent-plus-protease subvirion particles (dpSVPs) were produced by treating virions with chymotrypsin in the presence of micelle-forming concentrations of alkyl sulfate detergents. Infections with dpSVPs in murine L or canine MDCK cells provided evidence that the cleavage of mu1/mu1C during viral entry into these cells is dispensable for reovirus infection. Additionally, dpSVPs behaved like ISVPs in their capacity to permeabilize lipid bilayers and to undergo transcriptase activation in vitro, supporting the conclusion that cleavage of mu1/mu1C to mu1delta/delta and phi during viral entry is not required for either membrane penetration or transcriptase activation in cells. The capacity of alkyl sulfate detergents to inhibit the cleavage of mu1/mu1C in a reversible fashion suggests a specific association between virus particle and detergent micelles that may mimic virus particle-phospholipid membrane interactions during reovirus entry into cells.

Topics: Animals; Capsid; Capsid Proteins; Cattle; Cell Line; Chymotrypsin; Detergents; DNA-Directed RNA Polymerases; Dogs; Endopeptidases; Enzyme Activation; Hemolysis; In Vitro Techniques; L Cells; Mice; Micelles; Reoviridae; Sodium Dodecyl Sulfate; Virus Replication

Vibrio cholerae produces a cytolytic toxin named El Tor cytolysin/hemolysin which is encoded by the hlyA gene. This cytolysin is produced as a 79-kDa precursor form (pro-HlyA) into the culture supernatant after cleavage of the signal peptide of the hlyA product (prepro-HlyA). The pro-HlyA is then processed to a 65-kDa mature cytolysin (mature HlyA) after cleavage of the 15-kDa N-terminal peptide (pro region) of the 79-kDa precursor, usually at the bond between Ala-157 and Asn-158. We investigated whether proteases could process the recombinant 79-kDa pro-HlyA to the 65-kDa mature HlyA. We observed that the soluble hemagglutinin/ protease (HA/protease; a major protease of V. cholerae), trypsin, alpha-chymotrypsin, subtilisin BPN', papain, and thermolysin all processed the pro-HlyA to the 65-kDa mature form of the protein. Along with this, the protease-processed HlyA showed drastically increased hemolytic activity. The N-terminal amino acid of the mature form of cytolysin generated by HA/protease was Phe-151, and that due to trypsin was Ser-149. Other proteases also cleaved the pro-HlyA at a nearby site, between Leu-146 and Ser-153, and all the processed cytolysins showed increased hemolytic activity. These data suggest that the active El Tor cytolysin of V. cholerae could be derived from the C-terminal region of a pro-HlyA following proteolytic cleavage of the bonds in the vicinity of Leu-146 to Asn-158 by any of a wide variety of proteases.

Topics: Amino Acid Sequence; Bacterial Proteins; Chymotrypsin; Cytotoxins; Endopeptidases; Hemagglutinins; Hemolysin Proteins; Hemolysis; Metalloendopeptidases; Molecular Sequence Data; Papain; Protein Precursors; Protein Processing, Post-Translational; Subtilisins; Thermolysin; Trypsin; Vibrio cholerae

In order to understand the nature of cytolysin-membrane interactions, the characteristics of stable, non-lytic cytolysin-target cell intermediates formed at low ionic strength, neutral pH, and at physiological ionic strength, pH 6.0, were examined. Protease treatment of cytolysin-RBC intermediates formed at low ionic strength inhibited subsequent hemolysis when the intermediates were exposed to physiological ionic strength and pH. Similarly, when such intermediates were treated with anti-granule and anti-cytolysin antibodies a significant dose-dependent inhibition of hemolysis was observed. These results suggested that in this non-lytic state the cytolysin molecule was exposed on the RBC surface. If low ionic strength or pH 6.0 generated intermediates were washed in 0.5 M NaCl, hemolytic activity was greatly reduced and cytolysin activity could be recovered from the medium. In addition to RBC, both murine (Yac-1 and Lettre ascites) and human (K562) tumor targets formed cytolysin-target cell intermediates at low ionic strength and at low pH. Multilamellar vesicles composed of either phosphatidylcholine, sphingomyelin or phosphatidylserine inhibited the binding of cytolysin to RBC at both low ionic strength and pH 6.0 indicating a lack of polar head group specificity for cytolysin binding.

Topics: Animals; Cell Membrane; Chymotrypsin; Cytotoxins; Hemolysis; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Mice; Phosphatidylcholines; Phosphatidylserines; Pronase; Rats; Sodium Chloride; Sphingomyelins; Trypsin; Tumor Cells, Cultured

All-D-magainin-2 was synthesized to corroborate experimentally the notion that the biological function of a surface-active peptide stems primarily from its unique amphiphilic alpha-helical structure. Indeed, the peptide exhibited antibacterial potency nearly identical to that of the all-L-enantiomer. Being highly resistant to proteolysis and non-hemolytic all-D-magainin might have considerable therapeutic importance.

Topics: Amino Acid Sequence; Anti-Infective Agents; Antimicrobial Cationic Peptides; Chymotrypsin; Hemolysis; Humans; Indicators and Reagents; Magainins; Microbial Sensitivity Tests; Models, Molecular; Molecular Sequence Data; Peptide Fragments; Peptides; Protein Conformation; Stereoisomerism; Trypsin; Xenopus Proteins

A new potent inhibitor of complement system, named complestatin, was isolated from the mycelium of Streptomyces lavendulae SANK 60477. Complestatin (C61H45N7O15Cl6, MW 1,325) was a peptide compound having two unusual amino acids, D-(-)-4-hydroxyphenylglycine and D-(-)-3,5-dichloro-4-hydroxyphenylglycine. This compound inhibited the hemolysis of sensitized sheep erythrocytes (EA) mediated by guinea pig and human complement 50% at concentrations of 0.4 and 0.7 micrograms/ml, respectively, but did not trypsin and alpha-chymotrypsin activities at 200 micrograms/ml. When complestatin was administered intravenously to the sensitized guinea pigs, it strongly inhibited the systemic anaphylactic shock elicited by the antigen probably by blocking generation of anaphylatoxins (C3a and C5a).

Topics: Amino Acids; Anaphylaxis; Animals; Anti-Bacterial Agents; Chlorophenols; Chymotrypsin; Complement Activation; Complement Inactivator Proteins; Erythrocytes; Fermentation; Guinea Pigs; Hemolysis; Peptides, Cyclic; Streptomyces; Trypsin

The interaction of Sendai virus with small, unilamellar vesicles, lacking virus receptors and loaded with self-quenched 6-carboxyfluorescein, was studied. Sendai virions induced release of carboxyfluorescein from vesicles composed of negative charged phospholipids, despite the fact that they did not contain virus receptors. Preliminary experiments indicate that the carboxyfluorescein release is accompanied by mixing of the virus and liposome lipids and their entrapped contents, suggesting liposome-virus fusion. No release of carboxyfluorescein was observed with vesicles containing only phosphatidylcholine. The rate of virus-induced carboxyfluorescein release was temperature dependent; the lytic activity of the virus was greatly enhanced above 25 degrees C. This effect was not due to a thermal phase transition of the lipids in either the lipid vesicles or the virions. Virus-induced carboxyfluorescein release was inhibited by the presence of calcium ions in the medium and of cholesterol in the lipid vesicles. It increased with increasing concentrations of either the lipid vesicles or the virions. pretreatment of virions with increasing concentrations of three different proteolytic enzymes (trypsin, chymotrypsin and proteinase) inhibited the virus' ability to cause release of carboxyfluorescein from negatively charged liposomes. Inhibition of the viral lytic activity was also observed after virions were incubated above 56 degrees C.

Topics: Calcium; Chymotrypsin; Dithiothreitol; Electrochemistry; Endopeptidases; Fluoresceins; Hemolysis; Humans; Kinetics; Liposomes; Membrane Lipids; Parainfluenza Virus 1, Human; Phenylmethylsulfonyl Fluoride; Streptomyces; Temperature; Thermodynamics; Trypsin

Clathrin, a Mr = 72,000 clathrin-associated protein, and myosin were purified in milligram quantities from the same erythrocyte hemolysate fraction. Erythrocyte clathrin closely resembled brain clathrin in several respects: (a) both are triskelions as visualized by electron microscopy with arms 40 nm in length with globular ends and a flexible hinge region in the middle of each arm, and these triskelions assemble into polyhedral "cages" at appropriate pH and ionic strength; (b) both molecules contain heavy chains of Mr = 170,000 that are indistinguishable by two-dimensional maps of 125I-labeled peptides; and (c) both molecules contain light chains of Mr approximately 40,000 in a 1:1 molar ratio with the heavy chain. Erythrocyte clathrin is not identical to brain clathrin since antibody raised against the erythrocyte protein reacts better with erythrocyte clathrin than with brain clathrin and since brain clathrin contains two light chains resolved on sodium dodecyl sulfate gels while the light chain of erythrocyte clathrin migrates as a single band. The erythrocyte Mr = 72,000 clathrin-associated protein is closely related to a protein in brain that mediates ATP-dependent disassembly of clathrin from coated vesicles and binds tightly to clathrin triskelions (Schlossman, D. M., Schmid, S. L., Braell, W. A., and Rothman, J. E. (1984) J. Cell Biol. 99, 723-733). The erythrocyte and brain proteins have identical Mr on sodium dodecyl sulfate gels and identical maps of 125I-labeled peptides, share antigenic sites, and bind tightly to ATP immobilized on agarose. Clathrin and the uncoating protein are not restricted to reticulocytes since equivalent amounts of these proteins are present in whole erythrocyte populations and reticulocyte-depleted erythrocytes. Clathrin is present at 6,000 triskelions/cells, while the uncoating protein is in substantial excess at 250,000 copies/cell.

Topics: Carrier Proteins; Chymotrypsin; Clathrin; Electrophoresis, Polyacrylamide Gel; Hemolysis; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Macromolecular Substances; Microscopy, Electron; Molecular Weight; Myosins; Peptide Fragments

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.

Topics: Chlorpromazine; Chymotrypsin; Cold Temperature; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Glutaral; Hemolysis; Humans; Hydrogen-Ion Concentration; Hypotonic Solutions; Osmotic Fragility; Saline Solution, Hypertonic; Temperature

Although chymotrypsin treatment eliminated the reactivity of many antigenic determinants on the bovine red cell, haemolytic inhibition tests, double diffusion in agar tests, and immunizations provided no conclusive evidence of any of the affected determinants in the soluble digest. Chymotryptic digestion unmasked an apparent cryptantigen, rendering Q-positive cells reactive with a previously Q-negative anti-serum.

Topics: Animals; Blood Group Antigens; Cattle; Chymotrypsin; Epitopes; Hemolysis; Immunodiffusion; Rabbits

Treatment of the exterior surface of human erythrocytes with the proteolytic enzymes, trypsin or alpha-chymotrypsin (at 1 mg/ml), has no discernible effect on the carrier-mediated movement of glucose. However, the incorporation of either enzyme at much lower levels inside the erythrocyte by the method of reversible hemolysis leads to a progressive inhibition of the rate of glucose movement. Total inhibition eventually results at all tested concentrations of incorporated enzyme. These results strongly suggest that a protein susceptible to attack at the interior surface of the cell membrane is in some way involved in sugar transport. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the spectrin band (known to be located at the inner membrane surface) gradually disappeared during the protease treatment, in close parallel with the loss in glucose transport. This was not accompanied by any appreciable modification in Band III, which has been closely identified with the glucose transport system.

Topics: Anion Exchange Protein 1, Erythrocyte; Biological Transport; Cell Membrane; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Erythrocyte Membrane; Erythrocytes; Glucose; Hemolysis; Humans; Peptide Hydrolases; Time Factors; Trypsin

Topics: Animals; Cell Line; Chick Embryo; Chymotrypsin; Dose-Response Relationship, Drug; Fibrinolysin; Glycoproteins; Hemagglutination Inhibition Tests; Hemolysis; Molecular Weight; Mutation; Neutralization Tests; Pancreatic Elastase; Parainfluenza Virus 1, Human; Peptide Hydrolases; Pronase; Species Specificity; Thermolysin; Trypsin; Viral Plaque Assay; Viral Proteins; Virus Replication

A hemoglobin variant with the slightly fast mobility associated with K hemoglobins was found, together with hemoglobin S, in an apparently healthy 3-year-old Negro girl from Washington, D.C. Her father had the same variant, along with hemoglobin A, and her mother had hemoglobins A plus S. Respective proportions of hemoglobins K and S in the propositus were 66-34. The K variant now has been found to have the structural change Gln yields Glu at position beta-131 (beta-H9); therefore it apparently is identical to hemoglobin Camden reported in trait form in a Negro subject by YATES, BELLINGHAM and HUEHNS (Nature 243: 467-468, 1973).

Topics: Black People; Child, Preschool; Chymotrypsin; Electrophoresis, Starch Gel; Female; Glutamine; Hemoglobin, Sickle; Hemoglobinopathies; Hemoglobins, Abnormal; Hemolysis; Heterozygote; Humans; Peptides; Trypsin

Topics: Animals; Blood Group Antigens; Blood Proteins; Cattle; Cell Membrane; Chymotrypsin; Endopeptidases; Epitopes; Erythrocytes; Female; Hemolysis; Papain; Trypsin

Topics: Antigen-Antibody Complex; Autoradiography; Chymotrypsin; Complement System Proteins; Electrophoresis, Disc; Hemolysis; Humans; Immunodiffusion; Immunoelectrophoresis; Iodine Radioisotopes; Isotope Labeling; Microbial Collagenase; Ovalbumin; Pancreatic Elastase; Sodium Dodecyl Sulfate; Trypsin; Urea

Topics: Amino Acid Sequence; Animals; Biological Assay; Blood Glucose; Cattle; Chromatography, Ion Exchange; Chymotrypsin; Circular Dichroism; Diabetes Mellitus, Experimental; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Hemolysis; Hydrogen-Ion Concentration; Insulin; Insulin Antibodies; Iodine; Iodine Radioisotopes; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Radioimmunoassay; Seizures; Spectrophotometry, Ultraviolet; Sulfites; Tyrosine

Topics: Amino Acid Sequence; Amino Acids; Animals; Arginine; Carboxypeptidases; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Cyanogen Bromide; Electrophoresis, Paper; Guanidines; Hemolysis; Hydrolysis; Oxidation-Reduction; Proteins; Snakes; Spectrophotometry, Ultraviolet; Venoms

Topics: Amino Acids; Animals; Binding Sites; Caproates; Chymotrypsin; Complement Inactivator Proteins; Dialysis; Enzyme Activation; Esterases; Esters; Guanidines; Guinea Pigs; Hemolysis; Humans; Methylamines; Phenols; Rabbits; Sheep; Structure-Activity Relationship

Topics: Adult; Blood; Cell Membrane; Cell Wall; Chromatography, Gel; Chymotrypsin; Culture Techniques; Cytoplasm; Female; Hemolysis; Humans; Hydrochloric Acid; Infant, Newborn; Lectins; Lymphocyte Activation; Lymphocytes; Pepsin A; Pregnancy; Ribonucleases; Spectrophotometry; Streptococcus; Streptolysins; Thymidine; Tissue Extracts; Tritium; Trypsin; Umbilical Cord

Topics: Amidines; Chemistry Techniques, Analytical; Chymotrypsin; Depression, Chemical; Erythrocytes; Fibrinolysin; Hemolysis; Thrombin; Trypsin Inhibitors

Topics: Amino Acids; Animals; Chromosome Mapping; Chymotrypsin; Hemoglobins; Hemolysis; Peptides; Sheep; Species Specificity

The ability of streptolysin S preparations to induce high percentages of transformation in human peripheral blood lymphocytes was confirmed in a series of apparently healthy donors. Transforming activity was not demonstrated in the two media used for streptolysin S production, nor in control preparations in which a strain each of Streptococcus viridans, Staphylococcus aureus (nonhemolytic), and Diplococcus pneumoniae was substituted for the beta hemolytic streptococcal strain used for streptolysin S production. The relation of the hemolytic activity to the lymphocyte transforming activity of streptolysin S preparations was studied by means of inactivation and fractionation experiments. Heating produced a loss in both activities, but more in the hemolytic than in the transforming activity. The transformation obtained with a heated preparation had a high degree of correlation with that obtained with the unheated preparation in a series of normal subjects and patients with various rheumatic diseases, whose lymphocytes were often less responsive to stimulation with streptolysin S preparations (both heated and unheated) than the lymphocytes of the normal subjects studied. Treatment of streptolysin S preparations with chymotrypsin, vegetable lecithin, or trypan blue (the latter in minute amounts) resulted in preparations with no detectable hemolytic activity but with undiminished lymphocyte transforming activity. Chromatographic fractionations on DEAE-Sephadex columns yielded fractions endowed with transforming but not with hemolytic activity, and other fractions endowed with hemolytic but not with transforming activity. The recovery of the hemolytic activity was not complete and quantitation of the recovery of the transforming activity was not attempted. These experiments indicate that the hemolytic and transforming activities of streptolysin S preparations are independent of each other, and specifically that they are the attributes of two different streptococcal products, one of which is streptolysin S. The other is a nonhemolytic streptococcal product present in streptolysin S preparations but previously unrecognized. Some implications of these findings are discussed.

Topics: Blood Donors; Chromatography; Chymotrypsin; Hemolysis; Hot Temperature; Humans; Lymphocytes; Mitosis; Species Specificity; Staphylococcus; Streptococcus; Streptococcus pneumoniae; Streptolysins; Trypsin

Topics: Calcium; Carbon Dioxide; Chymotrypsin; Deoxyribonucleases; Escherichia coli; Hemolysin Proteins; Hemolysis; Oxygen; Ribonucleases; Strontium; Trypsin

Topics: Animals; Chemical Phenomena; Chemistry; Chymotrypsin; Complement Inactivator Proteins; Fluorides; Guinea Pigs; Hemolysin Proteins; Hemolysis; Pyridines; Sheep

Topics: Animals; Chymotrypsin; Complement Inactivator Proteins; Guinea Pigs; Hemolysin Proteins; Hemolysis; Pyridinium Compounds; Sheep

Topics: Animals; Calcium; Chemical Phenomena; Chemistry; Chymotrypsin; Detergents; Drug Stability; Edetic Acid; Erythrocytes; Hemolysis; Hydrogen-Ion Concentration; Phospholipases; Phospholipids; Phosphorus Isotopes; Rats; Serum Albumin; Solubility; Temperature; Trypsin

Topics: Animals; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Dextrans; Erythrocytes; Genetic Variation; Hemoglobins; Hemolysis; Molecular Biology; Peptides; Protein Hydrolysates; Sheep

Topics: Animals; Cattle; Cell Membrane; Chymotrypsin; Erythrocytes; Hemolysis; Kinetics; Papain; Peptides; Phospholipases; Radiation Effects; Rats; Serum Albumin, Bovine; Snakes; Spectrophotometry; Sulfhydryl Compounds; Sulfides; Trypsin; Venoms

Topics: Animals; Blood Coagulation; Blood Sedimentation; Brain; Chymotrypsin; Corticosterone; Dimethyl Sulfoxide; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Liver; Osmotic Fragility; Oxygen Consumption; Rats; Sulfur Isotopes; Trypsin; Urease

Topics: Antibodies; Cell Membrane; Chymotrypsin; Complement System Proteins; Erythrocytes; gamma-Globulins; Glucuronidase; Glycols; Hemolysis; Immunoglobulins; Lipase; Neuraminic Acids; Neuraminidase; Research; Trypsin

Topics: Acetylcholinesterase; Chymotrypsin; Endopeptidases; Erythrocytes; Hemolysis; Humans; Neuraminidase

Topics: Antitoxins; Cell Death; Chymotrypsin; Exotoxins; Hematologic Tests; Hemolysis; Pharmacology; Research; Staphylococcus; Toxins, Biological

Topics: Antitoxins; Bacterial Toxins; Cell Death; Chymotrypsin; Hematologic Tests; Hemolysis; Papain; Pharmacology; Research; Staphylococcus; Toxins, Biological; Trypsin