Showing papers on "Proteolytic enzymes published in 1971"

Carboxypeptidase A: a protein and an enzyme.

Abstract: Publisher Summary This chapter discusses the relationship of the three-dimensional structures of bovine carboxypeptidase A (CPA), and of its complexes with substrates and inhibitors, to the functional behavior of this enzyme. In particular, it discusses the basis for substrate specificity, modes of binding, and possible mechanisms of hydrolytic cleavage of substrates for this enzyme. CPA is a zinc-containing proteolytic enzyme, which catalyzes the hydrolysis of carboxy-terminal peptide bonds in protein and peptide substrates. Removal of Zn2+, either by lowering the pH below 5.5 or by the use of a variety of chelating agents at neutral pH, yields an inactive enzyme, apocarboxypeptidase A. Peptidase activity is known for Co2+, Ni2+, Mn2+ and Fe2+ in place of Zn2+, but substitution of Cu2+ for Zn2+ yields an enzyme that is inactive toward all substrates. Esters are also cleaved by CPA and the substitution of Hg2+, Cd2+, or Pb2+ retains esterase activity, although these heavy metal derivatives are not peptidases in solution. However, the crystals of the mercury derivative have shown some peptidase activity.

Creation of "amyloid" fibrils from Bence Jones proteins in vitro.

Abstract: "Amyloid" fibrils have been created from some human Bence Jones proteins by proteolytic digestion under physiologic conditions. These fibrils with an antiparallel, β-pleated sheet conformation consist of only a portion of the variable region of the immunoglobulin light polypeptide chain and share the physical properties of amyloid fibrils. The relation between amyloidosis and immunoglobulins is thus more firmly established and a pathogenetic mechanism for amyloid fibril formation is suggested.

Demonstration of two distinct components in the early antigen complex of Epstein-Barr virus-infected cells.

Abstract: Examination of numerous sera from patients with infectious mononucleodis (IM), Burkitt's lymphoma (BL) or nasopharyngeal carcinoma (NPC) for antibodieh to Epstein-Barr virus (EBV) induced early antigens (EA) revealed two distinct patterns of immunofluorescence in abortively EBV-infected Raji cells. One showed diguse (D) staining of the nucleus and cytoplasm of invaded cells, the other (R) was restricted to masses in the cytoplasm. Although D- or R-reactive Raji cells became detectable at similar times after exposure to EBV, the percentages of D-positive cells initially exceeded R-positive cells but ultimately both were nearly equal in number. R-positive cells almost invariably contained also D. In EBV-exposed RPM1 64–10 cells, frequently only D was synthesized. D antigen, in contrast to R, resisted fixation by methanol or ethanol, whereas R proved more resistant than D to proteolytic enzymes. Comparative serum titration on acetone, respectively ethanol-fixed Raji-EBV cell smears revealed that the transitory anti-EA response observed in many IM patients was restricted almost exclusively to anti-D. Anti-EA positive sera from NPC patients also showed dominantly anti-D activity whereas, in BL sera, anti-R was usually, but not always, dominant, often being the only antibody to the EA complex present. Preliminary tests with pronase-treated Raji-EBV cell smears indicated that dominantly anti-D reactive sera from IM patients were free of anti-R whereas such sera from NPC or BL patients usually gave positive reactions which in part, however, failed to conform with the R pattern. The possible implications of these results have been discussed.

The structure of papain.

Abstract: Publisher Summary The fruits of the tropical papaya tree have latex that contains several enzymes. This chapter focuses on two different proteolytic enzymes: chymopapain and papain. Both enzymes belong to the group of proteolytic plant enzymes that require a sulfhydryl group for activity. The chapter discusses the enzymatic properties of papain. Papain can catalyze a number of reaction—namely, acyl-enzyme intermediate, hydrolysis, transferase action, and specificity. X-ray diffraction can provide a wealth of information on a protein structure. The papain molecule consists of one folded polypeptide chain of 212 residues. The sequence determination of these residues is an example of the mutual interaction between purely chemical and X-ray methods. Because of the many difficulties that papain presented to the chemists, only a tentative sequence could be published in 1964. Some of the overlaps were not found to be as conclusive as desired, and at least one gap existed in the sequence. The available information, however, permitted the interpretation of the electron density map. Moreover, the difficulties in the early chemical sequence studies on papain because of the insolubility of the denatured protein and many of its peptides is circumvented by the use of completely carboxymethylated papain that has been fully maleylated.

The Nature of the Collagen Synthesized by Cultured Human Fibroblasts

Abstract: The hydroxyproline-containing proteins (hyproproteins) synthesized by cultured human fibroblasts have been partially characterized. The hyproprotein extracted from the cell layer was found to be similar to the collagen extracted from skin in the ratio of hydroxyproline to proline, chain composition, solubility, and resistance to proteolytic digestion. The hyproproteins isolated from the medium were different. About 20% of the peptide-bound hydroxyproline was found in randomly coiled chains. The α2 chains were present in considerable excess over the α1 chains, suggesting that the α2 chain may be synthesized in quantities greater than required to form a collagen molecule with a chain composition (α1)2α2. The remaining medium hyproprotein appeared to be an unusual form of native collagen which, unlike typical native collagen, was soluble under physiological conditions. This hyproprotein did not yield α chains when denatured and contained material that had a molecular weight greater than α chains. A similar size distribution was observed in the protein synthesized in the presence of β-aminopropionitrile, a specific inhibitor of collagen cross-linking. After treatment with pepsin, typical α1 and α2 chains were obtained from the protein in a 2:1 ratio. Since the medium protein is soluble and has properties different from the typical collagen molecule, it may represent a modified form that functions in the transport of collagen from the cell to the fiber.

Modification of methionyl-tRNA synthetase by proteolytic cleavage and properties of the trypsin-modified enzyme.

Abstract: Earlier studies have shown that native methionyl-tRNA synthetase from Escherichia coli K12 is composed of four, probably identical, subunits having a molecular weight of 43 000 each. Incubation of the purified enzyme with trypsin results in irreversible conversion into an enzymatically active modified form which, by a number of criteria, including molecular size, catalytic and antigenic properties, electrophoretic and chromatographic behaviour, appears to be undistinguishable from the altered methionyl-tRNA synthetase previously observed after incubation at 37°C of a crude extract. Furthermore, a similar modification of methionyl-tRNA synthetase resulted upon incubation of the purified enzyme with several other proteolytic enzymes, including papain, subtilisin and pronase. The structural and catalytic properties of the homogenous trypsin-modified methionyl-tRNA synthetase were examined in detail. It has a molecular weight of 64000 established by equilibrium ultracentrifugation, and dissociates in the presence of 8 M urea to yield two, apparently identical subunits of molecular weight 32000. Furthermore, it is shown that proteolysis of the native enzyme by trypsin is accompanied by release of enzymatically inactive fragments corresponding to approximately 20% of the original protein. The simplest interpretation of these results is that proteolysis selectively removes a portion of each original subunit, corresponding to a quarter of its size, with consequent dissociation of the original tetramer into dimers, each now composed of modified subunits. No significant differences in the Michaelis constants for any of the substrates or inhibitors tested were found between native and modified enzyme. Nor was conversion accompanied by any detectable change in the specificity towards the amino acid. The molecular activity (expressed as moles of substrate converted/mole subunit) of the homogenous trypsin-modified enzyme was found to be 20% lower than that of native methionyl-tRNA synthetase, for both reactions catalyzed by the enzyme. An interesting difference between the two forms of the enzyme appeared in their magnesium ion requirement for the aminoacylation of tRN As. Thus, under otherwise identical assay conditions, the trypsin-modified enzyme required significantly less magnesium ions for the aminoacylation of both tRNAMetf and tRNAMetm at optimal rates. Another remarkable feature of the trypsin-modified enzyme is its enhanced stability against heat inactivation, compared to the native enzyme. A structural model is proposed for methionyl-tRNA synthetase which attempts to account for its great susceptibility to limited proteolysis, and for the maintenance of its catalytic properties despite the resulting radical changes in its molecular structure.

20 Other Bacterial, Mold, and Yeast Proteases

Abstract: Publisher Summary The chapter discusses endopeptidases and focuses on their three main types: (1) acid proteases, (2) diisopropylphosphofluoridate (DFP)-sensitive alkaline proteases, and (3) metal chelator-sensitive neutral proteases. The acid proteases that include the Aspergillus saitoi acid protease, Rhizopus chinensis acid protease, and Paecilomyces varioti acid protease are characterized by low pH activity and stability profiles, insensitivity to metal chelating agents, phosphorylating compounds, such as DFP and thiol poisons. These enzymes show limited esterolytic activity and are reminiscent of the animal pepsins and rennins. The most widely distributed group of proteolytic enzymes of both microbial and animal origin is that of the the serine proteases, generally identified by their sensitivity to organophosphorus reagents, such as DFP and isopropylmethylphosphofluoridate. In addition to the three broad classes of endopeptidases, a number of different kinds of proteolytic enzymes are produced by bacteria and molds. Another unique group of proteases, which in some cases shares properties with one of the other groups, is the bacterial cell wall lysing proteases. These enzymes by virtue of the specific bonds cleaved have uniquely difierent characteristics from the other enzymes.

Protease Activities During the Course of Sporulation in Bacillus subtilis

Abstract: Three proteolytic enzymes have been isolated from sporulating cultures of Bacillus subtilis. These activities were, respectively, a protease inhibited by ethylenediaminetetraacetic acid (EDTA) but not phenylmethylsulfonyl fluoride (PMSF), a protease active on both protein and ester substrates, and an ester-active enzyme with low activity on proteins. The latter two enzymes were inhibited by PMSF but not by EDTA. The specific activity of each was determined both intra- and extra-cellularly during growth and sporulation in a single-defined medium. All three enzymes were shown to exhibit a rapid increase in specific activity at a time coinciding with the appearance of refractile bodies in cells.

Purification and Properties of Staphylococcal Delta Hemolysin

Abstract: Large amounts (200 mg per liter of culture supernatant fluid) of highly purified staphylococcal soluble delta hemolysin were obtained by adsorption to and selective elution from hydroxyapatite followed by exhaustive dialysis against water, concentration by polyvinylpyrrolidone or polyethylene glycol 20,000 dialysis, and a final water dialysis. No carbohydrate, phosphorus, or inactive 280-nm absorbing material was detected in the preparation; however, analysis by density gradient centrifugation, gel filtration, analytical ultracentrifugation, carboxymethyl cellulose chromatography, polyacrylamide disc gel electrophoresis, isoelectric focusing, and electron microscopy revealed that the lysin was molecularly heterogeneous. The preparation contained an acidic fibrous lysin (S20,w of 11.9) and a basic lysin component composed of a population of granular aggregates of various sizes, with a maximum S20,w of approximately 4.9. No other staphylococcal products were detected in the preparation. The lysin was active against erythrocytes from many animal species and acted synergistically with staphylococcal beta hemolysin against sheep erythrocytes. It was soluble in chloroform-methanol (2:1), was inactivated by various phospholipids, normal sera, and proteolytic enzymes, but was partially resistant to heat inactivation. Activity was not affected by Ca2+, Mg2+, citrate, ethylenediaminetetraacetic acid, or cysteine. The lysin preparation also disrupted bacterial protoplasts and spheroplasts, erythrocyte membranes, lysosomes, and lipid spherules, was growth-inhibitory for certain bacteria, and clarified egg yolk-agar. Large amounts produced dermonecrosis in rabbits and guinea pigs. The minimum lethal intravenous dose for mice and guinea pigs was approximately 110 and 30 mg/kg, respectively.

Selective differentiation between amyloid and connective tissue structures based on the collagen specific topo-optical staining reaction with congo red.

Abstract: There are some difficulties inherent in the identification of amyloid by Congo red anisotropy in the polarisation microscope, mainly because of the possibility of a nonspecific reaction of collageneous structures. The newly recognized collagen-specific Congo red topooptical staining reaction provides a theoretically well-founded possibility for differentiating between amyloid and collagen. In Canada balsam, or other apolar mounting media the Congo red anisotropy of amyloid as well as that of collagen are of the same (additive) optical sign. In gum arabic however they are of opposite signs, additive for amyloid and inversive for collagen. That difference enables a definite differentiation between amyloid and collagen, since in gum arabic Congo red stained amyloid is positively birefringent, and collagen is isotropic or weakly negatively birefringent. Pretreatment of the tissue sections with proteolytic enzymes resulted in a decrease in the general background staining of the tissues as well as in an increase of the inversive Congo red staining reaction of collagen and of the additive reaction of amyloid, providing thereby the possibility of differentiating sensitively and selectively between amyloid and collagen by Congo red anisotropy. Enzymatic removal of the elastic fibers prior to Congo red staining facilitated the exact localisation of senile amyloid deposits in the aortic wall. The initial amyloid deposits were visualized in the smooth muscle cells of the aortic wall. This finding may lead to a new approach to the pathogenesis of senile aortic amyloid.

Regulation and characterization of acid and alkaline phosphatase in yeast.

Abstract: SUMMARY: The activity of acid and alkaline phosphatase in baker's yeast is de-repressed when the organisms are starved of phosphate. Mutants lacking phosphatase activity and mutants with constitutive phosphatase synthesis were isolated. In all of them acid and alkaline phosphatases were affected simultaneously. Nevertheless, characterization of these two enzymes by sensitivity to inhibition by orthophosphate, temperature inactivation, proteolytic digestion and cation dependence showed that they were clearly distinct. It is suggested that these two enzymes share a common genetic and structural component.

14 Papain and Other Plant Sulfhydryl Proteolytic Enzymes

Abstract: Publisher Summary Papain is a simple protein containing only amino acids and devoid of carbohydrate All of the usual amino acids are present with the exception of methionine Papain contains no chromophoric groups other than its constituent amino acids It is rich in tyrosine and tryptophan The amount of active enzyme in papain solutions can be determined by a number of methods Finkle and Smith showed that the sulfhydryl (SH) titer of papain was a direct measure of the amount of active enzyme present Papain is routinely assayed in the presence of freshly prepared 0005 M cysteine and 0001 M ethylenediaminetetraacetic acid (EDTA) The mechanism of action of papain, ficin, chymopapain, and bromelain is very similar While the sequences near the essential thiol groups in these enzymes display varying degrees of homology, judgment as to whether all of these enzymes arose from a common evolutionary precursor has to await more extensive information on their amino acid sequences These enzymes are all activated by SH compounds and cyanide and inactivated by mild oxidizing agents

The essential amino-acid requirements of the prawnPalaemon serratus. The growth of prawns on diets containing proteins of different amino-acid compositions

Abstract: Two specimens ofPalaemon serratusPennant (of about 4 g wet weight) were each given intrahaemocoelic injections of [U-14C] acetate. They were maintained under controlled conditions, and the release of14CO2 and other labelled metabolites was followed for 6 days. The prawns were then sacrificed and fractionated into trichloroacetic acid soluble compounds, lipids, nucleic acids and a protein/chitin residue. Little or no14C had been incorporated into arginine, methionine, valine, threonine, isoleucine, leucine, lysine, histidine, phenylalanine and tryptophan. It was inferred thatP. serratus cannot synthesize these amino acids from ordinarily available materials, and that the prawns have an absolute dietary requirement for them. The glucosamine isolated from the protein/chitin residue contained more radioactivity than any of the other compounds examined. This implies a rapid turnover of glucosamine and chitin inP. serratus. The gain in weight of prawns fed on compound diets containing freeze-dried cod muscle was about 70% that of prawns fed on fresh mussel mantle. Predigestion of freeze-dried cod muscle with proteolytic enzymes did not significantly enhance its nutritional valve as assessed by increases in weight. Prawns fed two other compounded diets containing proteins of low nutritional value (gelatin and zein, respectively) grew at about 1/5 the rate of prawns fed fresh mussel mantle. Supplementation of the gelatin and zein in these diets with tryptophan, and with lysine plus tryptophan, respectively, failed to augment their nutritional value significantly.

3 Leucine Aminopeptidase and Other N-Terminal Exopeptidases

Abstract: Publisher Summary This chapter focuses on leucine aminopeptidase and other N-terminal exopeptidases. The classic N-terminal exopeptidase—leucine aminopeptidase—was first observed in the extracts of swine intestinal mucosa. Although several substrates could be used to assay leucine aminopeptidase, L-leucinamide is most often employed because it is commercially available or is easily prepared. It is rapidly hydrolyzed by leucine aminopeptidase, but it is not hydrolyzed by dipeptidases or by many other proteolytic enzymes that may be present at the various stages of purification of the enzyme. For the assay of leucine aminopeptidase during purification procedures, the titrimetric procedure as described by Hill et al . has been widely used because many samples may be conveniently assayed at one time. The highly purified preparations of leucine aminopeptidase were found to contain traces of contaminating endopeptidases, which could be inactivated by treating the preparations with diisopropyl fluorophosphate (DFP) and iodoacetate (SO). The inactivation of the contaminating enzymes is unnecessary when leucine aminopeptidase is used for complete hydrolysis of peptides.

“Disjunction” of Frog Neuromuscular Synapses by Treatment with Proteolytic Enzymes

Abstract: VERTEBRATE skeletal muscle fibres are surrounded by the ectolemma or basement membrane, a thin sheath of filamentous material. At the neuromuscular junction, the ectolemma occupies the synaptic cleft and is continuous with a similar material which surrounds the axon terminal and its Schwann cell covering1 (Fig. I A). Changes in the ectolemma of atrophic muscles have been observed2, 3, but little is known about the structure and function of this material. The study described here demonstrates that certain proteolytic enzymes selectively digest the ectolemmal sheath and that concomitantly the motor nerve terminals and their associated Schwann cells dissociate from the muscle fibres.

Alteration of Sites on the Mammalian Sperm Surface following Capacitation

Abstract: THE ovum cannot be penetrated by a spermatozoon until the latter undergoes a change termed capacitation1 which, in the golden hamster, can be produced in vitro by incubation in various,body fluids2–4 including serum5 and (β-glucuronidase6. The process involves vesiculation of the plasma membrane with the outer acrosomal membrane7, which in turn seems to release egg-penetrating enzymes and exposes a surface which can fuse with the vitelline membrane of the egg8. As part of a series of investigations on the nature of these surface changes we have studied the effect of proteolytic enzymes on the adherence of sperm to eggs in vitro. Our results show that adherence of sperm to the zona pellucida is species specific both before and after capacitation. Capacitation alters the sperm surface, however, so that it will no longer adhere to trypsin-treated eggs.

Sodium conductance activation without inactivation in pronase-perfused axons.

Abstract: A controversy of long standing in membrane electrophysio-logy is whether the sodium ion current (INa) and potassium ion current (IK) pass through the membrane in separate channels, or through a single set of channels which conduct first INa and then IK. In support of the latter hypothesis it has been noted that the sodium conductance (gNa) decline, called inactivation, proceeds with about the same time course as the potassium conductance (gK) increase. This could mean that Na+ selective channels are being converted into K+ selective channels. The hypothesis is especially interesting because of the possibility that the carrier postulated in active transport is convertible from Na+ to K+ selectivity1. An explicit statement of the single channel hypothesis and the means for disproving it were given by Mullins2. Because a single channel could not simultaneously conduct INa and IK, disproof requires that membrane conductance (gm) be made somehow to exceed the maximum value of gNa or gK. We report here that inactivation of gNa can be destroyed fairly selectively by the action from inside the axon of the unspecific proteolytic enzymes of pronase. In many cases gm after pronase treatment is greater than maximum gK before treatment, making untenable the single channel hypothesis.

Isolation and Some Properties of a Proteolytic Enzyme from Escherichia coli (Protease I)

Abstract: By the use of electrophoretic and spectrophotometric methods two types of endopeptidases were demonstrated in crude extracts of Escherichia coli: one was active on N-acetyl-dl-phenyl-alanine-2-naphtyl ester and the other on N-benzoyl-l-arginine-p-nitroanilide. The two activities were separated by gel filtration on Sephadex G-100. After gel electrophoresis of crude extracts, three bands of activity toward acetyl-phenylalanine-naphthyl ester were visualized. The enzyme corresponding to the band with the strongest esterolytic activity was also responsible for casein-hydrolytic activity in gel between pH 6 and 8; it was isolated and characterized. Starting with 450 g of wet cells, about 3 mg of a purified enzyme were obtained. This represented a recovery of 16% and almost 1000-fold purification. The isolated enzyme, designated as protease I, was homogeneous by electrophoresis and sucrose-gradient centrifugation. Its molecular weight was estimated by two different methods to be about 43000. The enzyme hydrolysed N-acetyl-dl-phenylalanine-2-naphthyl ester, a chymotrypsin substrate, but was inactive upon several synthetic substrates for enzymes with carboxypeptidase-A and trypsin-like specificity. The esterolytic activity was inhibited by DFP, whereas it was resistant to phenyl methyl-sulfonylfluoride even after prolonged treatment. Metal chelating agents, sulfhydryl reagents and several metal ions were without effect. The proterolytic activity of the purified enzyme was confirmed by its ability to breakdown native E. coli polynucleotide phosphorylase.

Kinetic specificity in papain-catalysed hydrolyses.

Abstract: The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.