Group-specific Inhibitors Research Articles

Protective effect of TAK242 blocking Toll-like receptor 4 pathway on septic myocardial injury and cardiac dysfunction

To investigate the role of Toll-like receptor 4 (TLR4) pathway on myocardial injury and cardiac dysfunction in septic rats. According to the random number table, 18 male Sprague-Dawley (SD) rats were divided into control group, lipopolysaccharide (LPS) group and TLR4 specific inhibitor TAK242 pretreatment group (TAK242+LPS group) with 6 rats in each group. The rat model of septic cardiac dysfunction was induced by intraperitoneal injection of LPS 15 mg/kg, and the control group was given the same amount of normal saline. The TAK242+LPS group was intraperitoneally given injection of TAK242 [it was injected intraperitoneally at a dose of 3 mg/kg and dissolved in 10% dimethyl sulfoxide (DMSO) and 90% corn oil according to the concentration of 0.2 g/L] 3 hours before LPS stimulation. The control group and LPS group were given the same amount of 10% DMSO and 90% corn oil. The cardiac function of rats in each group was examined by Doppler echocardiography 14 hours after injection of LPS. The blood of abdominal aorta was taken and the level of serum troponin (cTn) was measured by enzyme linked immunosorbent assay (ELISA). Myocardial tissue was harvested for hematoxylin-eosin (HE) staining, and the morphological changes of myocardial tissue were observed under light microscope. The mRNA expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in myocardial tissue were detected by real-time fluorescence quantitative polymerase chain reaction (qPCR). The protein expression of TLR4 in myocardial tissue was observed by immunohistochemical method. Western blotting was used to detect the levels of TLR4, nuclear factor-κB p65 (NF-κB p65) and its phosphorylation (p-NF-κB p65) in myocardial tissue. (1) The cardiac function and myocardial injury: Doppler echocardiography showed that the levels of left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) in the LPS group were significantly higher than those in the control group, while left ventricular ejection fraction (LVEF) and left ventricular shortened fraction (LVFS) were significantly lower than those in the control group. The degeneration, necrosis and inflammatory cell infiltration of cardiomyocytes were found with light microscope in the LPS group, and the levels of serum cTn were significantly higher than those in the control group, indicating that LPS-induced sepsis could cause cardiac dysfunction and myocardial injury. TAK242 blocking TLR4 pathway had a protective effect on cardiac function and myocardium during sepsis. LVEDV and LVESV in the TAK242+LPS group were significantly lower than those in the LPS group [LVEDV (μL): 71.25±21.16 vs. 118.01±11.89, LVESV (μL): 9.57±5.75 vs. 32.70±9.22, both P < 0.01]. LVEF and LVFS were significantly higher than those in the LPS group [LVEF: 0.868±0.075 vs. 0.722±0.095, LVFS: (59.88±8.46)% vs. (42.37±8.71)%, both P < 0.05]. Myocardial tissue injury was significantly reduced, and the serum cTn level was significantly lower than that in the LPS group (μg/L: 107.85±21.38 vs. 152.25±27.46, P < 0.05). (2) Inflammatory parameters: the results of qPCR, Western blotting and immunohistochemistry showed that the mRNA expressions of IL-6 and TNF-α, the expression of TLR4 protein and the p-NF-κB p65/NF-κB p65 ratio in the LPS group were significantly higher than those in the control group, indicating that LPS-induced sepsis could activate the inflammatory response mediated by TLR4/NF-κB pathway in the heart. However, blocking the TLR4 pathway by TAK242 could inhibit the TLR4/NF-κB pathway and reduce the myocardial inflammation in septic rats. The mRNA expressions of IL-6 and TNF-α, the expression of TLR4 protein and the p-NF-κB p65/NF-κB p65 ratio in the TAK242+LPS group were significantly lower than those in the LPS group [IL-6 mRNA (2-ΔΔCT): 10.44±3.30 vs. 107.50±29.48, TNF-α mRNA (2-ΔΔCT): 2.38±0.68 vs. 3.77±0.56, TLR4 protein (TLR4/GAPDH): 0.39±0.01 vs. 0.58±0.04, p-NF-κB p65/NF-κB p65 ratio: 1.21±0.11 vs. 2.10±0.18, all P < 0.05]. TAK242 can protect LPS-induced cardiac dysfunction and myocardial injury by blocking the TLR4 mediated inflammatory response.

Archaeal nitrification is a key driver of high nitrous oxide emissions from arctic peatlands

Bare peat surfaces created by frost action and wind erosion in permafrost peatlands have been shown to emit high amounts of nitrous oxide (N2O). With global warming, emissions of this highly potent greenhouse gas are expected to increase in Arctic permafrost peatlands. In natural unmanaged soils with low nitrogen deposition, such as Arctic soils, nitrification is the main source of nitrite and nitrate, and thus a key driver of N2O emissions. Here, we investigated nitrification, ammonia oxidizer populations and N2O production in vegetated and bare peat soils from four distant Arctic geographic locations. Through a combination of molecular analyses and group-specific inhibitor assays, we show that ammonia oxidation, the first step in nitrification, is mainly performed by ammonia-oxidizing archaea (AOA). All soils from different locations, including bare peat soils with high N2O emissions, harbored only two AOA phylotypes, including an organism closely related to Ca. Nitrosocosmicus spp. This indicates that high N2O emissions from these ecosystems are primarily fueled by nitrification mediated by very few archaeal species. To our knowledge, arctic peat soils in this study are the first natural environments where high N2O emissions have been linked to AOA. Any changes in archaeal nitrification induced by global warming will therefore impact on N2O emissions from the permafrost peatlands.

Reducing meat consumption: Identifying group-specific inhibitors using latent profile analysis

Consumption of animal products is an important greenhouse gas emitting behavior. However, perceived hindrances to incorporating more plant-based diets present challenges for the successful design of behavior-change interventions. Latent profile analysis of survey responses revealed three distinct groups. Meat-reducers perceive the fewest inhibitors and are the most willing to incorporate more meat-free days in their diets. Moderate-hindrance meat eaters perceive many more inhibitors, and are hindered by a lack of social support, attachment to meat, not wanting to change their routine, and less awareness of the health benefits of eating less meat. They are willing to incorporate new healthy foods in their diet and are somewhat willing to avoid meat on some days. Strong-hindrance meat eaters report weak self-efficacy and the most inhibitors but are somewhat willing to incorporate healthier foods in their diets. Implications for tailored meat-reduction interventions are discussed. For example, when targeting meat-attached individuals, it might be beneficial to focus on replacing red meats with less carbon-intensive protein sources.

The activity of nitrifying microorganisms in a high-altitude Andean wetland.

High-altitude wetland holds freshwater springs, evaporitic ponds and lagoon with variable salinity and nutrients, potentially influencing the ecology of nitrifying communities. In this study, nitrifying microorganisms in Salar de Huasco (Chile) were surveyed to determine bacterial and archaeal contribution to ammonium (AO), nitrite oxidation (NO), ammonium uptake (AU) during wet and dry seasons. The activity signals from these groups were assessed by specific amoA-qPCR transcription, 15N tracer studies and addition of group specific inhibitor experiments for nitrifying microorganisms (N1-guanyl-1, 7-diaminoheptane [GC7]-archaeal specific and allylthiourea [ATU]-bacterial specific). Nitrifying communities, i.e. Nitrosopumilus, Nitrosospira, Nitrosomonas, Kuenenia and Nitrospira, were more frequent (∼0.25% of 16S rRNA sequences) at low salinity sites. Bacterial amoA-qPCR transcripts also increased at low salinity and along in situ ammonium increase observed between wet/dry seasons. Nutrient changes through time and 15N tracer experiments results showed that AO and NO were detected and peaked mainly at low salinity-high ammonium sites (<37 000 μS cm-1 and >0.3 μM), whereas AU was predominant at evaporitic sites. Our results indicate that salinity and ammonium affect the nitrifying communities that are potentially more active at low-salinity sites but persistent at saltier evaporitic areas of the wetland when ammonium is available.

Correlation between heparanase and vascular endothelial growth factor in human retinal microvascular endothelial cells induced by hypoxia

Objective To investigate the effects of heparanase and vascular endothelial growth factor (VEGF) and their correlation in CoCl2-induced human retinal microvascular endothelial cells (HRECs) in an hypoxia model.Methods Human eyes were selected to establish CoCl2-induced HRECs hypoxia model in this study.Four experimental groups were studied:normal control group,hypoxia group (CoCl2 100 μmol/L,48 hours),PI-88 group (specific competitive inhibitor of heparanase: phosphomannopentaose sulfate,PI-88,5 μg/ml,combined with CoCl2 100 μmol/L,48 hours) and PBS control group.Heparanase,VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence.Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal,hypoxia,PI-88 and PBS control groups. Results Immunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs,but faint in normal group.Heparanase was also observed in the nucleus of hypoxia HRECs.Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group (Hpa:F=- 4.005,P ＜ 0.05 ; VEGF:F =- 4.063,P ＜ 0.05),and VEGF was decreased in HRECs treated with PI-88 (F=5.963,P＜0.05).Conclusions Heparanase is up-regulated that resulted in increase of VEGF expression,therefore enhanced angiogenesis in CoCl2-induced hypoxia HRECs. Key words: Retinal neovascularization/pathophysiology; Vascular endothelial growth factors; Anoxia; Retinal vessels; Endothelial cells

Polymorphisme protéique sanguin chez le poney de Mogod de Tunisie

Le poney de Mogod est originaire du nord ouest de la Tunisie. Ses aptitudes physiques lui confèrent une bonne adaptation et utilisation en montagne. Depuis le début du XXe siècle son effectif a subi une réduction considérable. Un plan de relance nécessite une meilleure caractérisation de la race, notamment d’un point de vue génétique. De plus, celle-ci permettrait de rechercher une éventuelle filiation de ce poney avec les autres chevaux de la région. Les systèmes électrophorétiques de l’albumine (Al), du composant GC (Gc), de la postalbumine (Xk), de l’estérase basique (Es), de la transferrine (Tf) et de la préalbumine (Pi) ont été étudiés chez 47 poneys de Mogod par la technique d’électrophorèse en gel de polyacrylamide. Les fréquences alléliques des six systèmes ont été estimées comme suit : AlA : 0,36 ; AlB : 0,64 ; GcF : 1,00 ; XkK : 1,00 ; EsI : 0,64 ; EsG : 0,18 ; EsF : 0,17 ; EsS : 0,01 ; TfD : 0,41 ; TfF2 : 0,31 ; TfH : 0,12 ; PiL : 0,35 ; PiU : 0,22 ; PiR ; PiS : 0,09. Une comparaison entre le polymorphisme protéique sanguin du poney de Mogod et celui des chevaux pur-sang Arabes, Barbes et Arabes-Barbes a été réalisée sur une base bibliographique.

Metalloproteinase Plays a Role in Mucin Secretion

Background : Mucus hypersecretion in the patients with airway diseases represents poor prognosis as well as discomfort. However, there is no known therapy for its effective control. One important component of mucus is mucin, a glycosylated protein, which endows mucus with viscosity. We studied whether a proteinase has a role in mucin secretion and if so, which. Methods : (1) Inhibition of mucin secretion Group-specific proteinase inhibitors were tested to evaluate whether a proteinase belonging to a group of proteinases plays a role in mucin secretion. Phenylmethylsulfonyl fluoride(PMSF, a serine proteinase inhibitor), E-64(a cysteine proteinase inhibitor), Pepstatin(an aspartic proteinase inhibitor) and 1, 10-Phenanthroline(a metalloproteinase inhibitor) were treated into the Calu-3 cell line for 24 hours. The enzyme linked immunoabsorbant assay(ELISA) for MUC5AC was performed to evaluate the amount of mucin secretion and to compare with a control. (2) Stimulation of mucin secretion Matrix metalloproteinase-9(MMP-9), MMP-12 and TACE(TNF-alpha converting enzyme), which are known to be related with airway diseases, were used to be treated into Calu-3 for 24 hours. ELISA for MUC5AC was performed to evaluate the amount of mucin secretion and to compare with the controls. Results : (1) PMSF(), E-64(), Pepstatin() and 1, 10-Phenanthroline() reduced the MUC5AC secretion by (meanstandard deviation; P=1.0 compared with the control), (P=0.34), (P=0.34) and (P=0.03), respectively. (2) The amounts of MUC5AC secretion stimulated by MMP-9(250ng/ml), MMP-12(100ng/ml) and TACE(200ng/ml) were (P=0.39), (P=1.0) and (P=0.39), respectively, compared with the controls. Conclusion : Metalloproteinase(s) is (are) suggested to play a role in mucin secretion. It appears that metalloproteinases, other than MMP-9, MMP-12 or TACE, affect the mucin secretion in this in vitro model.

Purification and Characterization of Phosphoglucomutase from Heterotrophic Tissues of Brassica campestris L

Two isoforms of phosphoglucomutase (PGM, EC 2.7.5.1) designated as PGM-I and PGM-II were purified from developing seeds of Brassica campestris L to their electrophoretic homogeneity. Both the forms had molecular mass of 59 kD each and were monomeric. PGM-I exhibited maximum activity at pH 7.5, while PGM-II evinced pH optima at 8.25. Both the forms exhibited hyperbolic response towards increasing concentrations of the substrate with Km values of 0.10 mM for PGM-I and 0.12 mM for PGM-II and had absolute requirement for glucose-1,6-P2. Fructose-1,6-P2 and 2,3-diphosphoglyceric acid inhibited the two forms non-competitively, whereas, ribulose-1,5-P2 inhibited only PGM-II, with Ki value of 0.8 mM. ATP inhibited the enzyme uncompetitively with Ki values for 0.26 mM (PGM-I) and 0.12 mM (PGM-11). Use of group specific protease inhibitors indicated Ser, His and Cys to play significant role in catalysis. On the basis of their differential behaviour and kinetic properties, PGM-I and PGM-II may be the forms present in cytosol and leucoplasts, respectively.

Biochemical characterization of pectate lyases produced by fluorescent pseudomonads associated with spoilage of fresh fruits and vegetables

An improved method for purification of pectate lyases (PLI and PLII) from culture fluids of Pseudomonas fluorescens CY091 and Ps. viridiflava PJ-08-6 by using a phosphocellulose cation exchanger was described. Analysis of purified PLI and PLII by sodium dodecyl sulphate-polyacrylamide and isoelectric focusing gel electrophoresis revealed that both enzymes had been purified to near homogeneity. Optimal Ca2+ concentration required for PLI and PLII activity was determined to be 0·5 mmol l−1. The Ca2+ requirement could not be replaced by other metal cations such as Mg2+, Cu2+, Zn2+, Fe3+ and Co2+. Optimal pH for activity was determined to be between 8·5 and 9·0. The Km values for sodium polygalacturonate were 1·28 and 1·11 mg ml−1 for PLI and PLII, respectively. Both PLI and PLII were stable at low temperatures (25°C or below) for at least 1 month. However, at 37°C, the activity decreased 50% in 36 h. Optimal temperatures for activity were estimated to be 46° and 52°C for PLI and PLII, respectively. Thermal stability of both enzymes at elevated temperatures (48°C or higher) increased when CaCl2 or a positively charged molecule such as polylysine was present, but decreased when polygalacturonate or a negatively charged molecule such as heparin was present. PLI and PLII exhibit differential degrees of sensitivity to group-specific inhibitors, including iodoacetic acid and diethylpyrocarbonate. This result suggests that both sulphydryl and imidazole groups are important for the catalytic function of PLI and PLII.

Variability of genetic serum protein markers in Austria

Haptoglobin (HP), transferrin (TF), group specific component (GC) and protease inhibitor (PI) polymorphisms were studied in a population sample (n=272) from Tyrol (Austria). Comparing the allele frequencies of these polymorphic serum protein systems with those reported on other Austrian population samples some heterogeneity in the regional distribution of allele frequencies was seen, especially concerning the GC system. The reasons for that may be due to different historical backgrounds in the various regions of Austria.

A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor.

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.

Genetic serum protein markers (HP, TF, GC, PI) in three population samples from Thuringia (Germany); Jena, Erfurt and Suhl

Haptoglobin (HP), transferrin (TF), group specific component (GC) and protease inhibitor (PI) polymorphisms were studied in three population samples from Thuringia: Jena, n=204; Erfurt, n=213; Suhl, n=180. GC and in particular TF allele frequencies show some statistically significant distribution heterogeneity, whereas HP and PI allele frequencies do not show any remarkable distribution differences. The results are discussed and compared with those obtained previously on other population samples from the eastern parts of Germany.

Characterization of xylose reductase from the yeast Pichia stipitis: Evidence for functional thiol and histidyl groups

Xylose reductase (EC 1.1.1.21) from the yeast Pichia stipitis NRC 2548 was purified to homogeneity via a two-step protocol using anion-exchange and gel-filtration chromatography. The pH-activity profile revealed the presence of two ionizable groups with pK app values of 5.8 and 8.1, suggesting the catalytic involvement of histidyl and thiol groups, respectively. Additional evidence supporting the involvement of these residues was provided by the use of group-specific inhibitors. The enzyme was rapidly inactivated in a pseudo-first order manner by the thiol-specific modifier p-chloromercuriphenylsulphonate (PMBS) and analysis of the order-of-reaction suggested that one essential cysteine residue was modified to effect inactivation. Treatment of the enzyme with another thiol-specific modifier, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), showed that modification of one cysteine per monomer led to 90% loss of activity, further supporting the existence of one essential cysteine residue. Inactivation by PMBS was reversed by adding 1 mM-β-mercaptoethanol. Inactivation of xylose reductase by the histidine-specific modifier diethylpyrocarbonate (DEP) followed a pseudo-first order process, and analysis of the order-of-reaction suggested that one essential histidine residue was modified to effect inactivation. Treatment of DEP-inactivated enzyme with 0.2 M-neutral hydroxylamine resulted in the recovery of 45% of enzyme activity. Protection of xylose reductase from PMBS- and DEP-inactivation was provided by NADPH and NADH but not by NADP+, D-xylose or DL-glyceraldehyde. This suggests that the essential histidine and cysteine residues may be involved with binding of cofactor by the P. stipitis xylose reductase.

Catalytic subunits of the multicatalytic proteinase

The multicatalytic proteinase (MCP) has been found in a wide variety of eukaryotic cells [1,2], and a similar enzyme has been identified in archaebacteria [ 3 ] . MCP is a high molecular mass (700 kDa) non-lysosomal proteinase which may be involved in both ubiquitin-dependent and ubiquitin-independent pathways of intracellular protein degradation. The proteinase complex isolated from rat liver seems to be composed of approximately 20 different components with molecular masses ranging from 22 to 34 kDa and isoelectric points ranging from 5 to 8.5 [41. The subunits appear by electron microscopy to be arranged in the shape of a hollow cylinder. The proteinase complex can cleave peptide bonds on the carboxyl side of basic, hydrophobic and acidic amino acid residues [5], and the proteolytic activities have been referred to as trypsin-like, chymotrypsin-like and peptidyl glutamyl peptide hydrolase activities, respectively. Results of studies with a variety of inhibitors have shown that these activities are catalysed at distinct sites. It is of interest to identify which components of the complex are responsible for each of the different proteolytic activities. In this study a method has been devised for selectively labelling MCP components responsible for the trypsin-like activity. The proteinase complex was isolated from fresh rat livers using a series of chromatographic steps [4]. The method of labelling involves use of a site-specific reversible inhibitor of MCP (leupeptin) and a group-specific irreversible inhibitor (N-ethylmaleimide) . Leupeptin inhibits serine and cysteine proteinases by forming a hemiacetal or thiohemiacetal with the catalytic serine or cysteine residue, respectively [7]. Leupeptin selectively inhibits the trypsin-like activity of the MCP complex in a competitive fashion at concentrations which have no effect on the other proteolytic activities. This inhibition can be reversed either by dialysis or by the addition of sodium borohydride to reduce the reactive aldehyde group. However, the recovery of activity by either of these methods was found to be slow. NEM is also an inhibitor of the trypsinlike activity of MCP [El. It also inhibits the peptidyl glutamyl peptide hydrolase activity of MCP but has little effect on the chymotrypsin-like activity. NEM is an irreversible and specific modifier of thiol groups at neutral pH, and is available in a [14C] -labelled form. Leupeptin and NEM were used together in a differential labelling protocol where MCP was first protected by leupeptin and then modified by NEM. Both inhibitors were used at concentrations which were known to fully inhibit the trypsin-like activity. Leupeptin was removed by dialysis and then [14C]NEM was added to label sites which were previously protected by leupeptin. An experiment was also carried out in the absence of leupeptin using only labelled NEM to establish how many thiol groups react with NEM. After dialysis to remove excess labelled NEM, polypeptide components of the two labelled proteinase preparations were separated by two-dimensional SDS polyacrylamide gel electrophoresis. Labelled component (s) were identified by fluorography of the dried gel. The results of these experiments showed that up to 28 moles of [l4C1NEM were incorporated in to each mole of unprotected MCP, whereas only approximately two moles of [I4C]NEM were incorporated per mole of MCP following the differential labelling protocol. These results imply protection by leupeptin of two thiol groups per MCP molecule. Fluorography of the two different labelled MCP preparations showed that almost every component of the complex can be labelled by NEM, but that in experiments designed to label only those NEM-reactive thiol groups protected by leupeptin, only one component is strongly labelled. These labelling experiments suggest that the trypsin-like activity is located in only one of the many different components of the proteinase complex. Recent results from the labelling of catalytic sites possessing the chymotrypsin-like activity also suggest that only one component is involved [9]. These findings support the view that MCP is a complex composed of distinct components which have different functions. This work was supported by the Medical Research Council. A.J.R. is a Lister Institute Research Fellow.

Proalbumin to Albumin Conversion by a Proinsulin Processing Endopeptidase of Insulin Secretory Granules

A lysate of purified insulin secretory granules, which contains two types of proinsulin processing activity (type 1, Arg-Arg-directed and type II, Lys-Arg-directed (Davidson, H.W., Rhodes, C.J., and Hutton, J. C. (1988) Nature 333, 93-96), was found to process proalbumin by specific proteolytic cleavage of the COOH-terminal side of the Arg-2-Arg-1 sequence. The subcellular distribution of proalbumin processing activity in insulinoma tissue paralleled that for proinsulin conversion and occurred principally in a secretory granule fraction. Cleavage appeared to result from the Arg-Arg-directed type 1 proinsulin processing endo-peptidase. It was Ca2+-dependent (K0.5 activation = 1.0-1.5 mM Ca2+), unaffected by group-specific inhibitors of serine, cysteinyl, or aspartyl proteinases, and had an acidic pH optimum (5.5). Active-site inhibitor studies showed this activity had a preference for dibasic over monobasic amino acid sequences and indicated that the sequence of the dibasic site was an important determinant of the susceptibility of the substrate to cleavage. The activity did not process the proalbumin Christchurch mutant (Arg-2-Arg-1 to Arg-2-Gln-1). It was inhibited by the variant alpha 1-antitrypsin Pittsburgh (Met358 to Arg358; K0.5 = 100 nM) but not by other related proteins normally co-secreted with albumin from hepatocytes, namely alpha 1-antitrypsin M, alpha 2-macroglobulin, or antithrombin III. The insulin secretory granule proalbumin processing activity was indistinguishable from a proalbumin endopeptidase reported in rat liver membranes and similar to the yeast KEX-2 protease. These findings suggest that a highly conserved set of proprotein endopeptidases exists, which are specific for a dibasic sequence but broadly specific for proprotein substrates. Such enzymic activities appear to be active within both the constitutive and regulated pathways of secretion. Intraorganellar Ca2+ and pH appear to play a key role in regulating their activities.

Uptake of L-lysine by a double mutant of Saccharomyces cerevisiae.

A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport. Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system. The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol. The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate). The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine. The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule. The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre. It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached. Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue.

Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.

The nature of the endoproteolytic activity involved in the post-translational processing of proinsulin has been investigated in rat insulinoma tissue. 125I-proinsulin was converted by lysed insulin-secretory granules into insulin via an intermediate form identified as des-dibasic-proinsulin. This activity co-localized with immunoreactive (endogenous) insulin and carboxypeptidase H upon subcellular fractionation of the tissue, indicating a secretory-granular location. Under optimized conditions, conversion was quantitative. Inhibitor studies demonstrated that processing occurred by a reaction sequence involving cleavage on the C-terminal side of the pairs of basic amino acids, with subsequent removal of the newly exposed basic residues by carboxypeptidase H. Endoproteolytic activity was abolished by EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not by 1,10-phenanthroline or by group-specific inhibitors of serine, thiol or acidic proteinases. Inhibition by EDTA and CDTA could be reversed by both Ca2+ and Zn2+, although the former appeared to be the ion of physiological importance. Addition of Ca2+ in the absence of chelators stimulated endoproteinase activity, with a maximal effect at 5 mM, a concentration consistent with the intragranular environment. Similarly the pH optimum of 5.5 coincides with the prevailing intragranular pH. Together these properties suggest that the Ca2+-dependent endopeptidase described here is involved in vivo in the proteolytic processing of proinsulin.

Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W.

Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.

Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10).

The nature and subcellular localization of the enzymic activities responsible for the production of the 20 kDa protein betagranin from its 100 kDa chromogranin-A-like precursor was investigated in transplantable insulinoma tissue. [35S]Methionine-labelled precursor was converted by lysed insulin-secretory granules into betagranin and one or more proteins of 47 kDa, via intermediates in the 60-65 kDa range. Lysosome-enriched fractions also processed the precursor, but not into the peptides found in vivo; other fractions, including those enriched in Golgi, were inactive. Conversion of the precursor by granules was quantitative and the products were stable. Inhibitor studies showed that processing occurred by initial endoproteolytic cleavage at sites marked by pairs of basic amino acids, followed by removal of these by carboxypeptidase H. The endopeptidase activity appeared to be a novel metalloenzyme, with a markedly acidic pH optimum (4.8-5). It was inhibited by alanyl-L-lysyl-L-arginyl chloromethane (K0.5 = 1.3 microM), but to a much lesser extent by inhibitor analogues of processing sites defined by single or unpaired basic amino acid residues, e.g. alanyl-L-norleucyl-L-arginylchloromethane (K0.5 greater than 100 microM), leupeptin (K0.5 = 150 microM) and antipain (K0.5 = 40 microM). p-Chloromercuribenzoate (K0.5 = 13 microM), Hg2+ (K0.5 = 16 microM), Zn2+ (K0.5 = 0.8 mM) and vanadate (K0.5 = 7 microM) also abolished activity, as did various anions (SCN- greater than I- greater than Cl- greater than SO4(2-). Group-specific inhibitors of serine, thiol and acidic endopeptidases were without effect. EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not 1,10-phenanthroline, abolished endoproteolytic activity. Several bivalent cations could restore activity after EDTA or CDTA inhibition, including Ca2+, Zn2+, Mn2+ and Sr2+; however, the ion of physiological importance appeared to be Ca2+ (K0.5 = 8 microM). The properties of the granule endopeptidase and its subcellular localization suggested that it is of importance in processing chromogranin A in the pancreatic beta-cell.

Intracellular activity of mycelial proteinases during fruit-body development in Flammulina velutipes

In the mycelium of Flammulina velutipes cultured on potato–glucose solution the concentration of water-soluble protein began to decrease steeply before fruit bodies formed, but the total amount of protein per culture decreased only when fruit bodies began to elongate rapidly. The level remained very low during rapid elongation. Proteinase activity per unit weight of mycelium against milk protein and azoalbumin decreased in parallel with the concentration of mycelial protein. Activity against azure blue – hide powder was low until the onset of rapid fruit-body elongation and reached a maximum later during that phase. Azoalbumin was hydrolyzed more actively than hide powder. Proteolytic activity per gram of mycelium against mycelial protein was almost the same before fruiting as during rapid fruit-body elongation. Specific activities per milligram protein against the three types of nonnative substrates increased in parallel to a maximum late during rapid elongation when protein concentration was lowest. Specific activity against native mycelial protein was also higher in extracts from older mycelia. Proteolysis was optimal near neutral pH and very low at pH 3.2. Tests with group-specific proteinase inhibitors showed that metallo-proteinases predominate in the mycelium. Serine, and to a lesser extent carboxyl and thiol proteinases, were also present. Neither endogenous inhibitors nor activators of proteolysis were detected.