The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.     

Preparation of biodegradable porous films for use as wound coverings

We studied the preparation of polymeric films formed from solutions of poly-3-hydroxybutyrate and poly-ε-caprolactone in chloroform and methylene chloride. A morphological study of film chips (electron microscopy) showed that solvent evaporation results in the formation of a heterogeneous structure with interpenetrating pores (1–20 μm). We proposed a new method for introducing the proteolytic enzyme and the aminopolysaccharide chitosan into the composition of polyester films. Composite films possessed necrolytic activity and were characterized by increased hydrophilicity. Properties of enzyme-containing films from a mixture of polymers (proteolytic activity, porous structure, and increased hydrophilicity) account for their use in the preparation of biodegradable wound coverings.     

Preparation of enzymes from raw materials of animal origin for medical application

The paper outlines the basic principles of production of enzymes for medical application from animal raw material. It presents complex scheme of the enzyme preparation from the pancreas of cattle and pigs, inhibitors of proteolytic enzymes from the pancreas and lungs of cattle as well as hyaluronidase action from the testes of cattle. The paper describes preparation scheme of purification of enzymes and protease inhibitors and results of their clinical study.     

Experimental study of a new biological preparation for treating E. coli infection and Proteus dysbacteriosis in young infants

The action of a new biological preparation intended for the treatment of E. coli infection and Proteus dysbacteriosis in children has been studied in experiments on mice and suckling rabbits. The protective activity of anti-E. coli and anti-Proteus lactoglobulin has been found to exceed that of normal lactoglobulin 6- to 13-fold. When subjected to the action of proteolytic enzymes of the gastrointestinal tract, the preparation retains its preventive properties on the level of the native preparation, which confirms the possibility of the oral administration of the preparation in clinical practice without protecting it from the proteolytic action of the enzymes of the gastrointestinal tract.     

Hymenolepis diminuta: interactions of the isolated brush border membrane with proteolytic enzymes

Proteins of the isolated brush border membrane of Hymenolepis diminuta were hydrolyzed in vitro by chymotrypsin, papain, pepsin, subtilopeptidase A (= subtilisin Carlsberg), and trypsin. Neither proteolytic nor amidase activity was demonstrable in the isolated membrane using proteinaceous (casein and hemoglobin) or chromogenic (benzoyl-arginine-p-nitroanilide and succinyl-alanyl-alanyl-propyl-phenylalanine p-nitroanilide) substrates, and the membrane preparation did not inhibit the proteolytic and amidase activities of these enzymes. Thus, the isolated tegumental membrane of H. diminuta is not inherently resistant to the action of proteolytic enzymes, and it does not inhibit proteolytic activity. In control incubations containing only buffer, the alkaline phosphatase activity of the brush border membrane decreased in a time dependent manner, but in the presence of chymotrypsin, subtilopeptidase A, and trypsin, the membrane retained greater alkaline phosphatase activity (pepsin and papain could not be tested for this effect on alkaline phosphatase activity). A similar time dependent decrease in activity was also noted for each of the proteolytic enzymes in control assays, but subtilopeptidase A and papain retained greater activity in the presence of the isolated membrane preparation when these assays were compared to controls.     

Inhibitory effect of antimicrobial preparation from lipids of marine fishes on tissue and microbial enzymes

We obtained a new food preservative from marine fish lipids possessing pronounced activity in relation to bacteria and microscopic fungi. The effects of this preparation on enzymes of microorganisms and muscle tissue of marine hydrobionts were studied. In vitro the preparation irreversibly inhibited acid and alkaline proteases and proteolytic and lipolytic enzymes of microorganisms and reduced enzyme activity in fish muscle tissue. The inhibitory effect of this preparation on enzymes contributes to stabilization of hydrolytic processes in meat of hydrobionts and suppresses microorganism growth in storage.     

Inhibitory Effect of an Antimicrobial Preparation from Lipids of Marine Fishes on Tissue and Microbial Enzymes

A new food preservative from marine fish lipids, was obtained possessing pronounced activity in relation to bacteria and microscopic fungi. The effects of this preparation on enzymes of microorganisms and muscle tissue of marine hydrobionts were studied. In vitro, the preparation irreversibly inhibited acid and alkaline proteases and proteolytic and lipolytic enzymes of microorganisms and reduced enzyme activity in fish muscle tissue. The inhibitory effect of this preparation on enzymes contributes to stabilization of hydrolytic processes in meat of hydrobionts and suppresses microorganism growth during storage.     

A method for the preparation of protease-free crystalline ribonuclease

A method is described for the preparation of crystalline ribonuclease free from all measurable traces of proteolytic enzymes.     

Activity of cytoplasmic proteinases from rat liver in Heren's carcinoma during tumor growth and treatment with medicinal herbs

The dynamics of the acid and neutral proteinases general enzymes activity change in the hepatocytes postnuclear fraction in the rats suffering from the Heren's carcinoma was investigated. It was determined that in the tumor development of the enzyme activity level of both the acid and neutral proteinases increased 2,6-fold. The natural preparation of the herbs (Calendula officinalis L., Echinacea purpurea L., Scorzonera humilis L., Aconitum moldavicum Hacq.) normalizes both the activity of the investigated enzymes and coefficients of the liver weights of the sick animals. The chemical medicinal preparation 5,6-benzcumarine-5-uracil normalizes the activity of the neutral cytoplasmatic proteinases and reduces the level of the proteolytic activity of the acid enzymes in comparison with the control group of the animals as well as increases of the liver weight coefficients.     

Comprehensive analysis of the N and C terminus of endogenous serum peptides reveals a highly conserved cleavage site pattern derived from proteolytic enzymes

The human serum proteome is closely associated with the state of the body. Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery. However, the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids, thereby limiting the clinical use of the endogenous peptides. We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics. The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions, including long-term incubation at 37°C and pretreatment with repeated freeze-thaw cycles. Furthermore, a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed. The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.     

The protease problem in Neurospora. Variable stability of enzymes in aromatic amino acid metabolism.

A proteolytic activity isolated from Neurospora crassa is shown to be responsible for the variable stability observed in vitro for enzymes involved in aromatic amino acid metabolism. For example, the activity of kynurenine formamidase was insensitive to the action of this protease preparation over a 24-h period of incubation at 25 °C, whereas chorismate synthase, anthranilate synthase, kynureninase, and the five activities of the arom multienzyme system were inactivated during this time. Anthranilate synthase and two of the arom system activities (dehydroquinate synthase and shikimate kinase) were inactivated by the protease preparation within 2 h. Phenylmethanesulfonylfluoride and a specific proteolytic inhibitor from N. crassa prevented inactivation of these enzymes. Spontaneous loss of activity at 25 °C of purified samples of anthranilate synthase, dehydroquinate synthase and shikimate kinase was also prevented by the inhibitors. A method for purifying the inhibitor from N. crassa is described, and its use as a reagent in the analysis of proteolytic action is demonstrated.     

Detection of proteolytic enzymes in agar electrophoresis

An agar substrate containing hyaluronic acid and horse serum for the detection of hyaluronidase activity following electrophoresis was found suitable for the detection of proteolysis. The substrate was refined for the detection of only proteolysis by elimination of the hyaluronic acid and substitution of bovine serum albumin in place of the serum. The refined substrate revealed two bands of proteolytic activity in papain. The refined substrate also revealed weak proteolytic activity in a “chromatographically pure” preparation of hyaluronidase. The method should be adaptable for the detection of multiple electrophoretic forms of several proteolytic enzymes.     

Electron Microscopy of Adenovirus 12 Replication 1. Fine Structural Changes in the Nucleus of Infected KB Cells

The ultrastructure of KB cells infected with oncogenic adenovirus 12 was studied at various intervals from 4 to 72 hr after viral inoculation. At 12 hr after infection, the nucleus and the nucleolus became hypertrophic. At 16 hr, bundles of fibers digestable by proteolytic enzymes were seen in the nucleus; they are considered as the early viral antigens identified immunologically by others. Between 24 and 26 hr, four types of nuclear inclusions appeared. Their sequence of appearance and fine structure are described. On the basis of their sensitivity to proteolytic digestion in thin sections, and the results of immunoferritin studies made by others, some of these inclusions are believed to represent viral structural antigens. Throughout the cycle of viral replication, the nucleolus displayed prominent and constant changes in the form of focal condensations and loosening of the nucleolonema, followed by atrophy and fragmentation. It is suggested that the early nucleolar changes reflect an active participation of the nucleolus in the synthesis of adenovirus 12. A hitherto unknown striated structure with definite periodicity, which is easily digested by proteolytic enzymes, was found in the nuclei during the late stages of adenovirus 12 replication.     

Purification and characterization of a bacteriocin from Klebsiella pneumoniae 158

Klebocin, a bacteriocin produced by Klebsiella pneumoniae 158, was purified to homogeneity by ammonium sulphate fractionation and sequential DEAE-Sephacel and Sephadex G-150 column chromatography. The purified preparation had an Mr of approximately 40 000 on SDS-PAGE. Chemical analysis of the purified preparation showed it to be a protein, and it was sensitive to digestion by various proteolytic enzymes.     

Preparation of pancreatin and purification of lipase from hog pancreas

Pancreatin containing high activities of proteolytic enzymes, amylase and lipase was prepared from optimally autolyzed hog pancreas. About one hundred grams of pancreatin were obtained from one kilogram of hog pancreas. Lipase was purified from the pancreatin preparation through steps of mild alkaline solution extraction, removing proteolytic enzymes by affinity adsorption, first ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and secondary ammonium sulfate fractionation. By these steps, the purity of the enzyme increased 14 fold and the recovery of the enzyme activity was 33%. The purified lipase was not homogeneous and contained several contaminating proteins when examined by disc polyacrylamide gel electrophoresis.     

Occurrence of Cell Lytic Enzymes in Blue-Green Bacteria

Detergent extracts of three blue-green bacteria (Agmenellum quadruplicatum strain BG1, Anacystis nidulans strain TX20, and Nostoc sp. strain MAC) contained enzymes capable of lysing suspensions of Micrococcus lysodeikticus. The enzyme preparation from A. quadruplicatum released soluble reducing fragments from purified peptidoglycan. The lytic activity exhibited a pH optimum between 6 and 7, was relatively heat stable, and was susceptible to attack by proteolytic enzymes. These results extend the range of bacterial types exhibiting cell lytic activity as well as confirm the existence of the lytic system commonly observed in "water blooms".     

A micromethod for determination of proteolytic enzymes in the pH range of 2.8 to 4.8. Radial enzyme diffusion into skim milk-containing agarose gel.

Radial diffusion of enzymes into a casein-containing gel is a fast and inexpensive technique for determination of proteolytic enzymes. A simple method for the preparation of substrate plates with a homogeneous distribution of caseinate and buffered at selected pH values between 2.8 and 4.8 has been described in this communication. By determination of pepsin in caseinate gels the accuracy was better than that described for photometric pepsin assays, and the sensitivity was 300 times greater.     

Proteinases of Streptomyces fradiae. I. Preliminary characterization and purification

A keratinolytic strain of S. fradiae has been shown to synthesize a complex of extracellular proteinases degrading native keratin proteins, elastin and collagen as well as some globular proteins. These enzymes are characterized by basic optimal pH and are inactivated by pheynlmethylsulfonyl fluoride (PMSF). Using preparative polyacrylamide gel electrophoresis, ion-exchange chromatography and affinity chromatography, 6 fractions of active protein of diversified proteolytic activity have been distinguished in the preparation studied.     

Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.     

The in-vivo and in-vitro biodegrading aggressiveness of Streptomyces fradiae enzymes

Highly active (2000 U g⁻¹) proteolytic enzymes were obtained from a partially purified, cell-free filtrate of a culture of Streptomyces fradiae. In vitro the enzyme was found to exhibit high activity as well as durability and thermostability (complete inactivation occurred only after 15 min at 120°C).The effects of the proteolytic action were confirmed in vivo in guinea-pigs. Within 30 min, an intracutaneously administered preparation caused drastic changes which were observable macroscopically. These extensive changes were established histopathologically.