Presence Of Proteolytic Activity Research Articles

695 Upregulated monocyte expression of PLIN2 is associated with plaque instability in coronary artery disease

Abstract Aims Perilipin 2 (PLIN2), a protein associated with intracellular lipid droplets (LDs), is involved in lipid metabolism of macrophages resident in atherosclerotic plaques and its up-regulation leads to LDs accumulation. LDs enlargement results in the macrophage transformation into foam cells, a key step for the onset of atherosclerosis. In the present study, we investigated the role of PLIN2 and its regulation mechanisms in atherosclerosis and plaque instability in patients with a diagnosis of ST-elevation myocardial infarction (STEMI) and stable chronic angina (SA). Methods and results We enrolled 120 patients with a diagnosis of STEMI and 42 SA patients with symptoms of stable effort angina lasting more than 12 months. Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA whole blood samples through standard gradient centrifugation over Ficoll-Hypaque. Monocytes were purified through indirect magnetic labelling of PBMCs. PLIN2 mRNA expression was investigated by Real Time-PCR and PLIN2 protein level was analysed in CD14+ monocytes by flow cytometry. Proteasome activity was assayed using AMC-tagged peptide substrate (Succ-LLVY-AMC), which releases free highly fluorescent AMC (Ex/Em 350/440 nm) in the presence of proteolytic activity. In CD14+ monocyte, PLIN2 protein expression was significantly increased in STEMI as compared to SA patients (P < 0.001), while PLIN2 mRNA level was not different in the two groups (P = n.s.). Despite proteasome activity was higher in STEMI as compared to SA patients (P < 0.001), significant inverse correlations were evident between PLIN2 levels and proteasome activity in the two groups (P = 0.05). Conclusions CD14+ monocyte PLIN2 protein expression was higher in STEMI as compared to SA patients suggesting an involvement in plaque instability. Despite proteasome activity was higher in STEMI patients, probably due to the elevated inflammatory burden, PLIN2 could escape proteasome degradation in a more efficient manner in STEMI as compared to SA patients.

Purification and biochemical characterization of fibrino(geno)lytic enzymes from tissues of Antarctic hydrobionts

Considering the continuing increase of morbidity and mortality rates associated with cardiovascular diseases, the search for novel compounds able to affect the hemostasis system is among the current trends of modern science and pharmacology. Fibrino(geno)lytic enzymes because of their role in dissolving blood clots as well as prevention of their formation attract special attention. The main goal of the current research was to develop the methodological approaches to obtain fibrino(geno)lytic enzymes from Antarctic hydrobionts and study their effects on the functioning of the hemostasis system. A complex approach which included affinity chromatography and size-exclusion chromatography was applied to isolate the fibrino(geno)lytic enzymes from the tissue of Antarctic nemertea (Parborlasia corrugatus), Antarctic sea urchin (Sterechinus neumayeri), and Antarctic sea star (Odontaster validus). The presence of proteolytic activity was monitored by zymographic technique. Fibrin(ogen)olytic activity was assessed by incubation of the samples with fibrinogen followed by 10% SDS-PAGE analysis. To test the substrate specificity of the enzymes, the chromogenic substrates such as H-D-Phe-Pip-Arg-pNA, pyroGlu-Pro-Arg-pNA, H-D-Val-Leu-Lys-pNA and Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA were used. The influence of fib rino(geno)lytic enzymes on platelet aggregation was assessed in platelet-rich plasma. To analyze the effect of the fibrino(geno)lytic enzymes on coagulation the blood coagulation time was assessed. The obtained results clearly indicated the presence of enzymes with activity toward fibrinogen in the tissues of tested hydrobionts. Based on the results of SDS-PAGE and zymography the molecular weight of the fibrino(geno)lytic enzymes was in the range of 26–34 kDa. The fibrinogen cleavage pattern analyzed by SDS-PAGE revealed the susceptibility of fibrinogen chains to degradation by enzymes from tissues of Antarctic hydrobionts. The fibrino(geno)lytic enzymes from all tested hydrobionts cleaved preferentially the Aα-chain and more slowly the Bβ-chain of fibrinogen. The fibrino(geno)lytic enzymes mediated the significant prolongation of blood clotting time in chronometric tests and inhibition of ADP-induced platelet aggregation. The enzymes exhibit activity against chromogenic substrates, which was more expressed in case of pyroGlu-Pro-Arg-pNA — a specific synthetic substrate for activated protein C and factor XIa. The enzymes isolated from the tissues of Antarctic marine hydrobionts possess a fibrin(ogen)olytic activity and can be of medical interest as therapeutic agents in the treatment and prevention of thrombotic disorders.

Estudio bioquímico del veneno de Tityus kaderkai(Scorpiones: Buthidae) con notas sobre su distribución y hábitat en el Perú

The biochemistry of the venom of Tityus kaderkai Kovařik, 2005 from Madre de Dios department, has been studied. The soluble venom contains 47.6% of protein. The venom proteins were separated from 12.9 mg of venom using cationic exchange chromatography in CM Sephadex C-25 with a 0.05 M ammonium acetate buffer pH 7.0. The chromatography profiles show seven peaks of proteins (I – VII) and five protein bands were distinguished in the crude venom, by PAGE-SDS. The toxicity assays allowed the identification of three toxins affecting Mus musculuswhich were associated to peaks IV, V and VII. Toxic proteins to Gryllus sp. were also found associated to peaks IV, V, VI and VII. Through the enzymatic activity, the presence of proteolytic activity over casein was found related to the first peak. Hyaluronidase activity has also been found in the peak IV with a specific activity 205.6 μg/min/mg. However, the crude venom and collected fractions did not show any phospholipase, anticoagulant, nor hemolytic activity. Notes on the distribution pattern and habitat are also included.

Protease activity, localization and inhibition in the human hair follicle

SynopsisObjectiveIn humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials.MethodsWe measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin.ResultsOur data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen® and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (UK, Brazil, China, first-generation Mexicans in the USA, Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system.ConclusionThese studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen® and climbazole. This technology may have potential to reduce excessive hair shedding.RésuméObjectifChez l'homme, le processus de perte de cheveux, désigné comme exogène, est censé se produire indépendamment des autres phases du cycle de cheveux. Bien que les mécanismes réels impliqués dans la perte de cheveux ne soient pas entièrement connus, il a été émis l'hypothèse que les processus conduisant à l'étape finale de la perte de cheveux peuvent être modulés par des protéases et/ou l'activité d'inhibiteurs de protéase. Dans cette étude, nous avons étudié la présence de protéases et de l'activité des protéases dans les cheveux humains perdus naturellement et évalué l'activité inhibitrice d'enzyme de différents matériaux.MéthodesNous avons mesuré l'activité enzymatique en utilisant un dosage basé sur la fluorescence et la localisation des protéines par immunohistochimie indirecte (IHC). Nous avons également développé un modèle de peau ex vivo pour mesurer la force nécessaire pour extraire les fibres capillaires de la peau.RésultatsNos données démontrent la présence d'une activité de la protéase dans le matériau de tissu entourant les racines du bulbe. Nous avons également démontré la localisation de l'expression des protéines de la sérine protéase spécifique du follicule pileux humain par IHC. Ces données fournissent des éléments de preuve démontrant la présence de protéases autour des racines du bulbe de cheveux qui peuvent jouer un rôle durant la phase exogène. Nous avons également testé l'hypothèse selon laquelle un nouveau système inhibiteur de protéase (combinaison de Trichogen ® et climbazole) pouvait inhiber l'activité de la protéase dans les extraits des bulbes de la racine des cheveux, recueillies à partir d'un éventail de groupes ethniques (Royaume-Uni, Brésil, Chine, 1ère génération Mexicains aux États-Unis, Thaïlande et Turquie) dans les deux sexes, mâles et les femelles. En outre, nous avons démontré que cette combinaison est capable d'augmenter la force nécessaire pour enlever les poils dans un système de modèle de peau ex vivo.ConclusionCes études indiquent la présence d'une activité protéolytique dans le tissu entourant le bulbe de la racine des cheveux humains et montrent qu'il est possible d'inhiber cette activité avec une combinaison de Trichogen ® et climbazole. Cette technologie peut avoir le potentiel de réduire la perte excessive de cheveux.

Proteolytic Activity in Actomyosinfrom Mantle and Fin of Squid (Illexargentinus) Stored at 2-4°C. Influence on the Physicochemical and Functional Properties of the Protein

<p>Actomyosin (AM) of mantle and fin from squid was stored at 2-4°C and the possible presence of proteolytic activity was investigated. Similar SDS-PAGE 10% patterns were obtained with both AM at zero time of storage. In absence of protease inhibitors, a decrease in the intensity of the band of the myosin heavy chain (MHC) and an increase in those of 155 kDa and 55 kDa bands of stored AM was observed. In presence of either PMSF (phenylmethylsulfonyl fluoride) or EDTA (ethylenediaminetetraacetic acid) both AM showed a minor degradation of the MHC, being the EDTA more effective. Proteolytic changes were accompanied by a significant increase (p < 0.05) in the proteinsurface hydrophobicity, (So: 1-anilino-8-naphtalene sulfonic acid (ANS)), the emulsion activity index (EAI) and the emulsion stability (ES) at 24 h of storage. At the same time, the reduced viscosity (VER) decreased significantly (p < 0.05). No significative changes (p > 0.05) in VER, IAE and ES of both AM occur in presence of protease inhibitors.A proteolytic activity closely associate with actomyosin from squid mantle and fin, was detected. Both serine and metalloproteinase activities were responsible for the autolysis of stored actomyosin. The best emulsifiying properties were obtained with AM without inhibitors. Because of this fins proteins could be and inexpensive source of functional ingredients with potential application as emulsifier agents in food product.</p>

Comparative analyses of proteolytic activities in seven species of synanthropic acaridid mites

Microplate assays with 96 wells were optimized to screen proteolytic activities in mite homogenates. Whole-mite extracts of Acarus siro, Aleuroglyphus ovatus, Tyrophagus putrescentiae, Tyroborus lini, Carpoglyphus lactis, Lepidoglyphus destructor, and Dermatophagoides farinae exhibited non-specific proteolytic activity in buffers from pH 2 to 12, and three peaks of highest activity at pH 3, 5-6, and 10 were distinguished. The reducing agent Tris(2-carboxyethyl)phosphine hydrochloride decreased general proteolytic activity on azocasein at pH 5 and 6. The results obtained on two non-specific substrates, azocasein and azoalbumin, showed highly different ranks of the species at pH 5 and 6. Proteolytic activities toward N(α)-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride, N-Succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-nitroanilide, N-Succinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide, Benzyloxycarbonyl-L-arginine-L-arginyl 4-nitroanilide, and N-Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-methionine 4-nitroanilide (MAAPMpNA) were highest at alkaline pH, but the activity toward MAAPMpNA was also high at pH 5 and 6. In contrast, N-Succinyl-L-alanyl-L-alanyl-L-phenylalanine 4-nitroanilide (AAPpNA) and L-arginyl 4-nitroanilide (ArgpNA) had the highest activity recorded at pH 6. The high activities observed on AAPpNA, ArgpNA, and MAAPMpNA at digestive pH suggest that enzymes present in these extracts could have the majority of proteolysis in the mite gut. Evidence of the presence of proteolytic activities on all tested substrates and in all the tested mite homogenates suggests that the proteolytic activities may contribute to allergenicity. Poor or undetected hydrolytic activities of mite extracts toward substrates for keratin and collagen at digestive pH underline the importance of ecological interactions between mites and microorganisms in the utilization of such substrates.

Frataxin degrading peptidase: A multifunctional regulator of mitochondrial energy production and iron balance

Frataxin is a conserved mitochondrial protein whose deficiency causes mitochondrial iron imbalance, energy deficit, and oxidative stress, leading to the progressive neurologic and cardiac disease Friedreich ataxia. We reported previously that the mature form of human frataxin is degraded to shorter products by an unknown protease, designated frataxin degrading peptidase (FDP). We hypothesized that FDP might represent a new mechanism to modulate mitochondrial iron balance via regulation of frataxin turnover. We have now purified FDP from different tissue sources. Interestingly, the enzyme corresponds to a known mitochondrial homodimeric oxidoreductase involved in energy metabolism and antioxidant defense. The presence of proteolytic activity has been confirmed with a recombinant form of the oxidoreductase produced in E. coli. The 3D structure of the enzyme suggests the presence of a Ser-His-Glu catalytic triad at the homodimer interface. Indeed, the proteolytic activity is stimulated by point mutations or other conditions that destabilize the homodimer, and is inactivated by certain serine protease inhibitors. Combining oxidoreductase and proteolytic functions in the same protein may be a mechanism to integrate changes in energy metabolism and redox status with mitochondrial iron balance. [Supported by the Muscular Dystrophy Association and a pre-doctoral fellowship from the American Heart Association (0415379Z)].

Effect of imported fire ant extract on the degradation of mountain cedar pollen allergen

Dust mite, cockroach, and mold extracts have been shown to contain proteases capable of degrading the proteins in other extracts. Loss of potency of allergens has been reported in mixtures containing cockroach and fungal extracts. Fire ant venoms consist of 90% to 95% n-alkyl and n-alkenyl piperidine alkaloids, which are not allergenic. No studies are available addressing the mixture of imported fire ant (IFA) whole-body extract with other allergens or the presence of proteolytic activity in the venom extract. To evaluate the stability of mountain cedar pollen extract mixed with IFA whole-body extract and to qualitatively analyze the extract mixture for degradation of mountain cedar protein. One milliliter each of mountain cedar and IFA whole-body extracts at a concentration of 500 microg/mL were combined and stored at 4 degrees C for 1, 3, 6, 15, 30, 60, 90, and 180 days. Separate mixtures of 1 mL of mountain cedar and IFA with 1 mL of human serum albumin were used as controls. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed, and protein bands were qualitatively analyzed for degradation. We detected 3 distinct IFA protein bands and 1 mountain cedar protein band. With respect to these bands, no protein degradation was observed during 6 months of study in the extract mixture compared with the controls. Imported fire ant whole-body extract does not seem to degrade mountain cedar protein. Mixtures of allergenic extracts may be able to include IFA whole-body extract.

Neurocrescin Is Specifically Cleaved after the Sequence DESD in a Caspase-3-Independent Manner

1. A neurite outgrowth factor, neurocrescin (NC), which we previously identified from an extract of denervated skeletal muscle, was endoproteolytically processed in cell transfectants. In addition to the processing site identified in NC (DESD358/F) being similar to the optimal recognition sequence of group II caspases, DExD, cleavage site mutations confirmed the involvement of caspase(s) in NC processing. 2. However, both the recombinant NC and the synthetic octapeptide (YLDESDFG) were scarcely cleaved in vitro by caspase-3 or -7. Furthermore, transiently expressed NC was cleaved even in the caspase-3-deficient cell line, MCF-7 cells, and this efficiency was not altered by the transfectional expression of caspase-3. 3. Using the fluorescent substrate (Ac-DESD-AMC), the characteristic proteolytic activities, which cleaved it more effectively than caspase-3 and whose pH dependences were different from those of caspase-3, were endogenously identified in the muscle extract. These findings indicate the presence of proteolytic activities that are distinguishable from caspase-3.

Proteolytic properties of Funastrum clausum latex

As part of a screening of latex endopeptidases from plants growing in Argentina, the presence of proteolytic activity in the latex of Funastrum clausum stems is reported. The proteases present in the crude extract showed the main characteristics of the cysteine proteolytic class, i.e. optimum pH at alkaline range, isoelectric point (pI) higher than 9.0, and inhibition of proteolytic activity by thiol blocking reagents. A remarkable thermal stability was also evident in the crude extract. Endosterolytic preference tried on p-nitrophenyl esters of N-α-carbobenzoxy- l-amino acids was higher for the alanine, asparagine and tyrosine derivatives. Preliminary peptidase purification by two-step ionic exchange showed the presence of two proteolytic fractions with molecular masses of approximately 24.0 kDa according to SDS-PAGE.

DETECCIÓN DE INHIBIDORES DE PROTEASAS EN EXTRACTOS DE LEGUMINOSAS Y SU EFECTO SOBRE PROTEASAS ENDÓGENAS DEL MÚSCULO DE PESCADODETECTION OF PROTEASE INHIBITORS IN LEGUMINOSE EXTRACTS AND ITS EFFECT ON ENDOGENOUS PROTEASES FROM FISH MUSCLEDETECCIÓN DE INHIBIDORES DE PROTEASAS NOS EXTRACTOS DE LEGUMINOSAS E O SEU EFECTO SOBRE PROTEASAS ENDÓXENAS DO MÚSCULO DOS PEIXES

The Modori phenomenon occurs in fish species from the Gulf of Mexico. The origin seems to be associated with serine and cisteine-proteases. The objective of this work was to determine the presence of protease inhibitors in crude extracts obtained from commercial leguminoses and to establish its capability to inhibit the proteolytic activity in surimi. The fish species employed were sole (Cyclopsetta chitendenni) and croaker (Micropogon undulatus). The leguminose extracts were obtained from kidney bean, pea, chickpea and soy. The presence of inhibitors in the extracts was confirmed employed trypsin, chymotrypsin and papain (1 g/L), having azocasein as substrate. The textural degradation in surimi gels was determined as changes in shear strength and shear strain (test of torsion). The presence of proteolytic activity at 60°C in surimi as well as the inhibitory effect of leguminose extracts were determined measuring the presence of soluble peptides in 5% trichloroacetic acid. The leguminose extracts contain inhibitors of serine-proteases for trypsine and chymotrypsine-like enzyme. Both species showed disminution in mechanic properties at 60°C, associated with proteolisis. Leguminose extracts inhibited the proteolytic activity in vitro. Protease inhibitors contained in crude legumonise extracts could be employed for inhibiting the proteolytic activity in fish products. Palabras clave: Modori, Protease inhibitors, Leguminoses. Resumen El fenómeno Modori está presente en especies de pescado del Golfo de México. El origen de este fenómeno parece estar asociado con serín y cisteín-proteasas. El objetivo de este trabajo fue determinar la presencia de inhibidores de proteasas en extractos crudos de leguminosas comerciales y establecer su capacidad de inhibir la actividad proteolítica en surimi. Las especies de pescado empleadas fueron lenguado (Cyclopsetta chitendenni)y croca (Mycropogon undulatus). Los extractos de leguminosas fueron obtenidos de alubia, chícharo, garbanzo y soja. La presencia de inhibidores en los extractos fue confirmada empleando tripsina, quimotripsina y papaína (1 g/L), teniendo azocaseína como sustrato. La degradación textural en geles de surimi se determinó como cambios en fuerza y deformación al corte (prueba de torsión). La presencia de actividad proteolítica a 60°C en surimi así como el efecto inhibitorio de los extractos de leguminosas se determinó midiendo la presencia de péptidos solubles en TCA al 5%. Los extractos de leguminosas presentaron inhibidores de serín-proteasas para enzimas tipo tripsina y quimotripsina. Ambas especies presentaron disminución en sus propiedades mecánicas a 60°C, asociada con proteólisis. Los extractos de leguminosas inhibieron la actividad proteolítica. Los inhibidores de proteasas presentes en los extractos crudos de leguminosas podrían emplearse para inhibir la actividad proteolítica en productos de pescado. Resumo O fenómeno Modori está presente en especies de peixe do Golfo de México. A orixe deste fenómeno parece estar asociado cas serín e cisteín-proteasas. O obxectivo deste traballo foi determiñar a presencia de inhibidores de proteasas en extractos crudos de leguminosas comerciais, e estabrecer a súa capacidade de inhibir a actividade proteolítica en surimi. As especies empregadas foron linguado (Cyclopsetta chitendenni) e croca (Mycropogon undulatus). Os extractos das leguminosas foron obtidos de alubia, chícharo, garbanzo e soja. A presencia de inhibidores nos extractos foi confirmada empregando tripsina, quimotripsina e papaina (1 g/L), tendo azocaseina como sustrato. A degradación textural en xeles de surimi determiñóuse como cambios en forza e deformación ó corte (probas de torsión). A presencia de actividade proteolítica a 60°C en surimi, asi como o efecto inhibitorio dos extractos de leguminosas determiñóuse medindo a presencia de péptidos solubles en TCA ó 5%. Os extractos de leguminosas presentaron inhibidores de serín-proteasas para enzimas tipo tripsina e quimotripsina. Ambas especies presentaron disminución nas súas propiedades mecánicas a 60°C, asociada con proteólisis. Os extractos de leguminosas inhibiron a actividade proteolítica. Os inhibidores de proteasas presentes nos extractos crudos de leguminosas poderían empregarse para inhibir a actividade proteolítica en productos obtidos a partir do peixe. Palabras clave: Modori, Inhidores de proteasas, leguminosas.

Carboxypeptidase Activity of the Zinc Metalloprotease in the Cement Precursor Secretion of the Barnacle, Chthamalus fragilis Darwin

The liquid cement precursor secretion (CPS) of the adult barnacle, Chthamalus fragilis, exhibits Zn-metalloprotease activity. To assess the bond specificity of the Zn-metalloprotease, samples of liquid CPS were collected by glass micropipette from the exposed bases of adult barnacles and incubated in 50 mM Tris buffer, pH 8.0, containing 10 mM Ca++, at 27°C or 20°C for 6 or 24 hr. in the presence of fluorescent dye-labeled synthetic C-1 and A-1 PepTag peptides. These peptides have a net positive charge and migrate toward the anode during agarose gel electrophoresis. When incubated with the C-1 PepTag peptide, CPS samples generated F1 hydrolysis fragments that failed to migrate during electrophoresis in 0.8% agarose gels, indicating the presence of proteolytic activity in the CPS. Proteolysis of the C-1 peptide was inhibited by 2.0 mM orthophenanthroline in the presence of 10 mM Ca++ ions. No hydrolysis products were generated when samples of CPS were incubated in the presence of the A-1 PepTag peptide. This suggested that the CPS contained a Zn-metalloprotease that exhibited a preference for the carboxy-terminal lysine of the C-1 PepTag peptide. There were no indications of aminopeptidase or endopeptidase activities in the barnacle CPS. When incubated with hippuryl-lysine and hippuryl-arginine substrates, samples of CPS gave activities ranging from 111 to 319 μmol hippuric acid formed/min/mg of CPS protein. These observations indicate that the CPS of C. fragilis contains a Zn-metallo-exoprotease with a preference for carboxy-terminal basic amino acids.

Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries.

There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.

Isolation and Preliminary Characterization of Extracellular Proteases Produced by Strains ofXylella fastidiosafrom Grapevines

Virulent and weakly virulent grape strains of Xylella fastidiosa grew well on PD3 amended with gelatin and produced zones of hydrolysis on this medium, indicating the presence of proteolytic activity. Proteases also were produced in PD3 broth and degraded gelatin, azocasein, and azocoll, Gelatin was the best substrate for the proteases. In addition to using the gelatin assay, native activity and sodium dodecyl sulfate activity gels aided in visualizing and monitoring protease activity. Strain P of X. fastidiosa produced at least two proteases, designated P1 and P2, of 54 and 50 kDa, respectively. Protease activity was most abundant in the 31-60% ammonium sulfate fractions [...]

Two site-specific radioimmunoassays which demonstrate the presence of proteolytically modified insulin-like growth factor-binding protein-3 in the circulation

The presence of proteolytic activity in the circulation directed against insulin-like growth factor binding protein-3 (IGFBP-3) was originally described in pregnancy but has subsequently been described in a number of catabolic and other pathological conditions. However, detection of this proteolytically modified IGFBP-3 has been demonstrated, in most cases, following separation using SDS-PAGE and this has led to speculation that its occurrence may be an artefact of the harsh conditions employed. Using two site-specific antisera, one of which recognizes as its antigenic site a region of IGFBP-3 which is close to that of the IGF-binding domain, we have developed two radioimmunoassays which, when compared, can reveal alterations in the IGF-binding domain of IGFBP-3. The presence of IGFBP-3 modified by the circulating protease is denoted in this system by a divergence in the IGFBP-3 levels observed; this divergence has been shown to coincide with protease activity as determined by Western ligand blotting. The use of these assays has confirmed that the IGF-binding site of IGFBP-3 undergoes proteolytic modification in the circulation during a number of pathologies.

Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.

Proteolytic activities in the developing seeds of ×Haynaldoticum sardoum

Aminopeptidase, carboxypeptidase and proteinase activities were measured in endosperms from unripe and ripe seeds of ×Haynaldoticum sardoum. Aminopeptidase and proteinase activities were high during the early maturation stages and then decreased. In contrast, carboxypeptidase activity increased with maturation. Localization studies demonstrated that aminopeptidase and carboxypeptidase activities were present in the three tissues examined (pericarp, green layer plus aleurone, and starchy endosperm). Proteinase activity against gliadin was located in the pericarp and in the green layer plus aleurone, but was absent in the starchy endosperm. The presence of proteolytic activities in the outer kernel layers might be correlated to the hydrolysis of transitory protein reserves during the senescence of the seed coatś. Aminopeptidase and carboxypeptidase activities located in the starchy endosperm could participate in the breakdown of protein reserves during the early phases of seed germination.

Fungal parasitism of the cyst nematodeHeterodera schachtii: infection and enzymatic activity

Abstract Fungal egg parasites isolated from eggs of the cyst nematode Heterodera avenae in Sweden were investigated with respect to their ability to infect cyst nematode eggs of H. schachtii in vitro. The infection was studied by interference phase contrast microscopy of whole cysts and of cryosections of cysts exposed to the fungi on agar plates. Verticillium suchlasporium was the most effective parasite, infecting 53% of the nematode eggs, while V. chlamydosporium infected 12% of the eggs. The fungi Paecilomyces lilacinus, Cylindrocarpon destructans or Fusarium oxysporum did not parasitize nematode eggs; nor did Arthrobotrys oligospora , a nematode trapping fungus nor Penicillium viridicatum which served as a control fungus. The ability of the fungi to infect eggs was correlated with their lytic enzyme activity. Fungi that readily infected eggs also showed chitinase activity and presence of proteolytic activity. The Verticillium species had an activity between 3.7 and 14.6 μmol N -acetyl-glucosamine per mg protein per hour (CU) while it was 4.5 CU or lower for P. lilacinus . Other isolates did not shown any chitinase activity.

Fungal parasitism of the cyst nematode Heterodera schachtii: infection and enzymatic activity

Fungal egg parasites isolated from eggs of the cyst nematode Heterodera avenae in Sweden were investigated with respect to their ability to infect cyst nematode eggs of H. schachtii in vitro. The infection was studied by interference phase contrast microscopy of whole cysts and of cryosections of cysts exposed to the fungi on agar plates. Verticillium suchlasporium was the most effective parasite, infecting 53% of the nematode eggs, while V. chlamydosporium infected 12% of the eggs. The fungi Paecilomyces lilacinus, Cylindrocarpon destructans or Fusarium oxysporum did not parasitize nematode eggs; nor did Arthrobotrys oligospora, a nematode trapping fungus nor Penicillium viridicatum which served as a control fungus. The ability of the fungi to infect eggs was correlated with their lytic enzyme activity. Fungi that readily infected eggs also showed chitinase activity and presence of proteolytic activity. The Verticillium species had an activity between 3.7 and 14.6 μmol N-acetyl-glucosamine per mg protein per hour (CU) while it was 4.5 CU or lower for P. lilacinus. Other isolates did not shown any chitinase activity.

Proteolytic activity in the insulin receptor

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.