The interaction of protein substrates (antigens) during anin vitro degradation by rabbit cathepsin D

The rate of degradation of human serum albumin (HuSA) by rabbit cathepsin D, measured on the basis of the amount of degradation products soluble in trichloroacetic acid, was shown to depend on the original concentration of the substrate and on the presence of other proteins in the system. Both human and rabbit IgG decreased the rate of cathepsin degradation of HuSA, whereas bovine haemoglobin, on the contrary, increased the rate of degradation. Immunochemical analysis (i.e. immunoelectrophoresis and radial immunodiffusion) have shown that the mixture of two proteins was degraded at a slower rate than either of these proteins alone. This phenomenon was studied using even a substrate possessing an enzymatic activity (catalase) and it was shown that the presence of a further substrate in the mixture decreased the degree of inactivation of catalase by cathepsin D. It suggests that competition between individual proteins and their fragments takes place in the course of the proteolytic reaction and, in addition, their association also occurs. Therefore the kinetics of catheptic degradation of a mixture of protein antigens is rather complicated. These results are discussed in view of antigen degradation within the cells and mechanism of killing of phagocytosed microorganisms.     

In situ protamine release: A versatile sample preparation method for the electrophoretic analysis of nuclear proteins on acid/urea-based gels

Protamine sulfate is used to release histones and other basic proteins from the DNA of chromatin. This phenomenon becomes the basis of a versatile method for analysis of the nucleic acid and protein composition of nucleoprotein samples, which is termed here in situ protamine release. When protamine is added to a nucleoprotein sample in 5% acetic acid and 8 m urea, at a concentration of 1.0%, ≥94% of the histones are released from the DNA of chromatin, comparable to the release of histones using sodium dodecyl sulfate. This makes in situ protamine release the method of choice for the analysis of acid-soluble proteins on acid/urea-based gels, where the DNA must be removed from the protein prior to electrophoresis. Compared to DNase I release or acid extraction, protamine release is found to be the simplest, most reliable, and most effective method for removing the acid-soluble proteins from DNA. Protamine is either added to the sample (very much like the detergent, sodium dodecyl sulfate), or electrophoresed through a gel containing nucleoprotein, thus displacing proteins in its path. A serendipitous advantage of protamine is that it can also serve as a carrier for the precipitation of dilute nucleoprotein samples with ethanol, and 3 mm Mg²⁺, to concentrate the nucleoprotein in preparation for analysis. A unique feature of the in situ protamine-release method is that the DNA is not lost or destroyed and can therefore be used for subsequent analysis.     

Enzymatic degradation of and bovine serum albumin release from starch-acetate films

The effect of acetylation of potato starch on swelling, enzymatic degradation, and bovine serum albumin (BSA, molecular mass 68 kDa) release rate from polymer films was studied. Potato starch and potato starch acetates (SA), having a degree of substitution of 1.9 or 2.6, were investigated. Polymer films were incubated in phosphate buffer solution pH 7.4 in the absence and presence of enzymes (alpha-amylase, amyloglucosidase, esterase) or in human serum. The acetylation of potato starch decreased its swelling considerably. Increased acetylation of starch also considerably retarded its enzymatic degradation. Due to the decreased swelling and degradation of SA films, BSA was released much slower from SA films than from potato starch films, both in the presence and absence of enzymes.     

Immunological evidence for a P2 protamine precursor in mature rat sperm.

High molecular weight proteins in Rattus norvegicus that are immunoreactive with an anti-protamine 2 specific antibody but not with an anti-protamine 1 specific antibody are described. These proteins were detected by coupling high-performance liquid chromatography (HPLC) with an enzyme-linked immunosorbent assay (ELISA). Briefly, following HPLC separation of rat sperm nuclear proteins, the HPLC fractions were probed with the antibodies. We estimate that the antibody probes are 100-1000 times more sensitive than UV absorbance measurements. Immunoblot analysis following acid-urea electrophoretic separation of rat sperm nuclear proteins, and of the HPLC fractions, also detected putative protamine 2 precursor proteins. The proteins reactive with the anti-protamine 2 antibody are most likely not mature protamine 2, since they were detected in a region of the chromatogram where we would not expect protamine 2 to migrate based on the chromatographic locations of human and mouse protamine 2. Likewise, the immunoblotting experiments demonstrated that the anti-protamine 2 antibody recognized proteins with slower electrophoretic mobilities than would be expected for a mature protamine 2. An anti-protamine 1 monoclonal antibody, Hup1N, that binds rat protamine 1 is also described. Hup1N allowed for identification of the HPLC fractions that contained rat protamine 1. Finally, we demonstrated that Hup1N binds protamine 1 from a large number of species, suggesting a conserved epitope for Hup1N.     

Protein Kinase and Specific Phosphate Acceptor Proteins Associated with Vaccinia Virus Cores

Incubation of purified vaccinia virus with gamma-(32)P-adenosine triphosphate resulted in the incorporation of (32)P into hot trichloroacetic acid-insoluble material. Enzymatic activity was completely dependent on the addition of divalent cations and was stimulated by nonionic detergents and dithiothreitol. Chemical studies demonstrated that serine and threonine residues of 15,000 molecular weight viral polypeptides were phosphorylated. In contrast, the major structural proteins were not phosphorylated or were phosphorylated to a much lesser extent. Added histones and protamine, but not serum albumin, casein, or phosvitin were phosphorylated by the partially disrupted vaccinia virus preparations. The protein kinase was tightly associated with vaccinia virus particles since the specific enzymatic activity remained constant during the final steps of virus purification, the specific activities of many different preparations of virus were similar, and the enzymatic activity cosedimented with vaccinia virus during rate zonal sucrose gradient and potassium tartrate gradient equilibrium centrifugations. Controlled degradation of vaccinia virus, with nonionic detergents and dithiothreitol, indicated that both the protein kinase and the specific phosphate acceptor proteins were located in the virus core.     

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.     

Inhibition of the enzymatic activity of thrombin by concanavalin A

Concanavalin A, a carbohydrate lectin derived from the jack bean, prolongs the thrombin clotting time of human plasma or purified fibrinogen. Prolongation is due to delay in peptide release from fibrinogen. The rate of fibrin monomer polymerization is not affected. Hydrolysis of protamine sulfate by thrombin is inhibited by concanavalin A. All inhibitory effects are prevented by α-methyl-D-mannoside. Concanavalin A does not delay clotting of fibrinogen by reptilase (releases fibrinopeptide A only) or by Ancistrodon contortrix contortrix (releases fibrinopeptide B initially followed by a small amount of A). It is concluded that concanavalin A binds to a carbohydrate on the thrombin molecule thus inhibiting its enzymatic activity.     

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte- mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.     

Lipase-catalyzed biodegradation of poly(epsilon-caprolactone) blended with various polylactide-based polymers

Poly(epsilon-caprolactone) was blended with various polylactide-based polymers and processed to films by the solution casting method. Blends of 25/75, 50/50, 75/25, 90/10, and 95/5 (w/w) poly(epsilon-caprolactone)/poly(l-lactide), a 95/5 (w/w) blend of poly(epsilon-caprolactone) with a poly(d-lactide), a 50/50 (w/w) poly(l-lactide)-poly(d-lactide) mixture, and a poly(l-lactide-co-epsilon-caprolactone) copolymer were considered comparatively. The various phase-separated films were allowed to degrade in the presence of Pseudomonas lipase, biodegradation being monitored by proton nuclear magnetic resonance, size exclusion chromatography, differential scanning calorimetry, X-ray diffraction, and environmental scanning electron microscopy. The formation of separated phases during solvent evaporation and their morphologies are discussed. The introduction of poly(l-lactide) dramatically decreased the degradation rate of poly(epsilon-caprolactone)/poly(l-lactide) blends. The higher the percentage of poly(l-lactide), the slower the degradation, while the presence of cracks and increasing the lipase concentration acted in favor of the enzymatic degradation. Long-term enzymatic degradation of the various 95/5 blends was investigated over 480 h. The poly(epsilon-caprolactone) phase was enzymatically degraded by the lipase regardless of the blend type, the degradation rate depending on the nature of the co-components.     

Oxidative insult in sheep red blood cells induced by T-butyl hydroperoxide: the roles of glutathione and glutathione peroxidase

Three different types of red blood cells (RBC) were used: (i) RBC from sheep having genetically high GSH (ii) RBC from sheep with genetically low GSH and (iii) RBC from high-GSH sheep treated with CDNB to deplete GSH. Incubation of these RBC with t-butyl hydroperoxide (tBHP, 3 mM) for 10 min caused the formation of TBARS, oxidation of haemoglobin and degradation and aggregation of membrane proteins in RBC from low-GSH sheep and GSH-depleted RBC. By contrast, RBC from high-GSH sheep (normal RBC) did not show the degradation and aggregation of membrane proteins within the first 10 min. Dithiothreitol (DTT) was highly effective in preventing the tBHP-mediated oxidation of haemoglobin, the formation of TBARS and the degradation and aggregation of membrane proteins in both normal RBC and low-GSH RBC. However, DTT did not provide protection in GSH-depleted RBC or normal RBCs in the presence of 1.5 mM mercaptosuccinate (MCS), a potent inhibitor of GSH peroxidase (GSHPx). The ability of GSH to prevent the oxidation of haemoglobin and the degradation and aggregation of membrane proteins was abolished in the presence of MCS. These results indicate that the protective function of DTT involves a GSH-dependent mechanism. Both GSH and GSHPx play key roles in this enzymatic system. In the light of the complete protection of RBC against oxidation induced by tBHP in the presence of DTT or GSH, the GSH/GSHPx system appears to act directly as a tBHP scavenger. The activities of four well-known antioxidants, Butylated hydroxytoluene, ascorbate, alpha-tocopherol and desferrioxamine were also tested in this study to cast further light on the role of free radical scavenging in protection from tBHP mediated free radical insult.     

Enzyme-responsive release of encapsulated proteins from biodegradable hollow capsules

Biodegradable hollow capsules encapsulating proteins were prepared via layer-by-layer assembly of chitosan and dextran sulfate on protein-entrapping mesoporous silica particles and the subsequent removal of the silica. The enzymatic degradation of the capsules in the presence of chitosanase was explored by scanning electron microscopy (SEM). With increasing time, the chitosan component was degraded by chitosanase, and the capsules began to deform and were finally destroyed. Sustained release of the encapsulated proteins was attained by using the enzymatic degradation of the hollow capsules. The release behavior was successfully manipulated by altering the charge of capsule surface.     

Role of the calpain system in muscle growth.

Muscle protein degradation has an important role in rate of muscle growth. It has been difficult to develop procedures for measuring rate of muscle protein degradation in living animals, and most studies have used in vitro systems and muscle strips to determine rate of protein degradation. The relationship between results obtained by using muscle strips and rate of muscle protein turnover in living animals is unclear because these strips are in negative nitrogen balance and often develop hypoxic cores. Also, rate of protein degradation is usually estimated by release of labeled amino acids, which reflects an average rate of degradation of all cellular proteins and does not distinguish between rates of degradation of different groups of proteins such as the sarcoplasmic and the myofibrillar proteins in muscle. A number of studies have suggested that the calpain system initiates turnover of myofibrillar proteins, which are the major group of proteins in striated muscle, by making specific cleavages that release thick and thin filaments from the surface of the myofibril and large polypeptide fragments from some of the other myofibrillar proteins. The calpains do not degrade myofibrillar proteins to small peptides or to amino acids, and they cause no bulk degradation of sarcoplasmic proteins. Hence, the calpains are not directly responsible for release of amino acids during muscle protein turnover. Activity of the calpains in living cells is regulated by calpastatin and Ca2+, but the nature of this regulation is still unclear.     

Determination in vitro of the rate of protein synthesis and degradation in human-skeletal-muscle tissue.

The present studies were aimed to evaluate the possibility to use a system for estimation in vitro of the biosynthesis and degradation rates of human skeletal muscle protein. A previously characterized human skeletal muscle preparation was used. Amino acids and insulin stimulated significantly the incorporation rate of leucine into proteins. The effect of amino acids was more pronounced than that of insulin. The stimulatory effect of insulin could be decreased by amino acids. Insulin did not influence the tissue uptake or the oxidation rate of leucine. The release of [14C]leucine deriving from degradation of prelabelled skeletal muscle fibre proteins was linear for at least 2.5 h of incubation and optimal with leucine at concentrations beyond 12.5 mmol/1 or in the presence of puromycin in the incubation medium. The rate of the release of radioactivity was significantly inhibited by amino acids and at borderline significance by insulin but not by puromycin. The specific radioactivity in prelabelled proteins decreased significantly in the presence of puromycin suggesting that leucine derived from protein degradation was reutilized in vitro. This reutilization was found to be 9 +/- 1% of leucine released from degradation of proteins in 30 subjects. A statistically significant positive correlation between the cathepsin D activity in human skeletal muscle tissue and the degradative rate of prelabelled muscle proteins in vitro was observed. The results indicate that biosynthesis and degradation of skeletal muscle proteins in this system in vitro were subjected to control mechanisms. It is suggested that the release of radioactivity from prelabelled muscle fibre proteins during incubation probably only reflects the degradation of some rapidly-turning-over proteins.     

A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

Control Mechanisms Operative in a Natural Microbial Population Selected for Its Ability to Degrade L-Lysine. III. Effects of Carbohydrates in Continuous-Flow Systems Under Shock Load Conditions

Two naturally selected microbial populations were maintained under continuousflow conditions with glucose or magnesium growth-limiting. The reactors were subjected to shock loads by changing the influent substrate from L-lysine to a mixture of L-lysine and glucose, L-lysine and fructose, or L-lysine and ribose. During the subsequent transient state, the following parameters were examined: lysine chemical oxygen demand (COD), carbohydrate COD, total COD, biological solids concentration, cell protein, enzymatic capability (lysine-degrading enzymes), and the rate of lysine removal. The carbohydrate was then removed from the influent and the same parameters were examined until a new steady state was established. In all cases, glucose and fructose caused a significant repression of the synthesis of lysine-degrading enzymes, resulting in a decrease in the enzymatic capability of the cells. In the carbon-limited reactor, the faster the flow rate, the greater was the repression, whereas, in the magnesium-limited reactor, the slower the flow rate, the greater was the repression. The introduction of ribose into the reactors caused an initial increase in lysine enzymatic capability followed by a slight repression when ribose degradation started.     

Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by peroxides

Hydrogen peroxide and organic hydroperoxides react with haemoglobin to release iron which can be complexed to apotransferrin, bleomycin and desferrioxamine. This released iron promotes deoxyribose degradation by a Fenton reaction, DNA degradation in the presence of bleomycin and stimulates lipid peroxidation. It is likely that iron released from haemoglobin is the true generator of hydroxyl radicals in the Fenton reaction.     

Sequence- and species-dependence of proteasomal processivity

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome's ability to unfold its substrate, and polyglutamine repeats such as found in Huntington's disease reduce the processivity of the proteasome in a length-dependent manner.     

Contrasting response of protein degradation to starvation and insulin as measured by release of N tau-methylhistidine or phenylalanine from the perfused rat heart.

An isotope-dilution method is described for the measurement of N tau-methylhistidine release from the perfused rat heart. We argue that release of N tau-methylhistidine is indicative of cardiac actin degradation. N tau-Methylhistidine release is compared with phenylalanine release in the presence of cycloheximide (phenylalanine release being a measure of degradation of mixed proteins). In hearts perfused with glucose plus acetate, the rate of actin degradation was increased by starvation and was not inhibited by insulin. In contrast, the rate of mixed-protein degradation was decreased by starvation and was inhibited by insulin. The fractional rate of degradation of mixed proteins in hearts from fed or starved rats was greater than that for actin. It is suggested that there are at least two pools of intracellular protein, the degradation rates of which differ in terms of their response to insulin and starvation.     

Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines

A soluble glycosaminoglycan sulfotransferase (3'-phosphoadenylylsulfate:chondroitin 4'-sulfotransferase, EC 2.8.2.5) from chick embryo cartilage has been prepared free from endogenous acceptor. The reaction with this enzyme preparation was stimulated by basic proteins and polyamines, the degree of stimulation being dependent on the chemical nature of both basic compounds and acceptor glycosaminoglycans. A maximum stimulation was obtained when protamine (basic compound) and chondroitin (acceptor) were involved in the reaction mixture at a molar ratio of protamine to repeating disaccharide units of chondroitin, 1:100. The stimulation of sulfotransferase activity by basic substances was much higher than that by Mn2+. However, increasing the Mn2+ concentration immediately reduced the stimulation by basic substances. The Km value for 3'-phosphoadenosine 5'-phosphosulfate of the sulfotransferase, when chondroitin was used as acceptor, was 1 . 10(-6) M in the presence of 25 microgram/ml protamine, compared to 2 . 10(-5) M in the absence of protamine. These observations indicate that the basic proteins and polyamines may interact with acceptor polysaccharide, thereby causing an increase in the affinity of the enzyme toward 3'-phosphoadenosine 5'-phosphosulfate.     

Electrochemical assay of plasminogen activators in plasma using polyion-sensitive membrane electrode detection

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these "clot-busting" drugs.