Cytotoxic, antioxidative, and ACE inhibiting activities of dolsan leaf mustard juice (DLMJ) treated with lactic acid bacteria

This study was performed to know whether there is any change of physiological activity in DLMJ which is inoculated by lactic acid bacteria. Lactic acid bacteria were isolated from Dolsan leaf mustard Kimchi (DLMK) at 20°C. In the optimum ripening period, the population ofLeuconostoc andLactobacilli in the DLMK were found to be high. TheLeuconostoc, Lactobacilli andLactococci strains were identified asLeuconostoc mesenteroides., Leuconostoc gelidum, Weissella confusa, Lactobacillus plantarum, Lactobacillus raffinolactis, Lactococcus lactis andWeissella confusa using the Biolog system. The most predominant strain which was isolated from DLMK wasWeissella confusa. As the results of the phylogenetic analysis using 16s rDNA sequence, theWeissella confusa turned out to beWeissella kimchii, with 99.0% similarity. To investigated the change of physiological activity in DLMJ by lactic acid bacteria, 7 predominant strains inoculated to DLMJ (Dolsan Leaf Mustard Juice). The cytotoxicity was found to be under 19.55% all cases Also, the antioxidative activity of the DLMJ treated with lactic acid bacteria was very low, which might have been due to the reduced antioxidative phytochemicals during the preparation of the sterile, sample. The ACE inhibiting activity of DLMJ by inoculation withWeissella kimchii was shown to be the highest (94.0%). This could be that the degradation of sulfur containing materials in DLMJ byWeissella kimchii gave rise to ACE inhibiting activity.     

Responses of seminal vesicle and testicular beta-glucuronidase and beta-N-acetylglucosaminidase to testosterone and some metabolites in Heteropneustes fossilis (Bloch).

Intraperitoneal administration of testosterone for 20 days produced differential effects on beta-glucuronidase and beta-N-acetylglucosaminidase (beta-Glc) activity in seminal vesicle (SV) and testis of the catfish Heteropneustes fossilis in preparatory phase (March). The lower dosages of 0.25 and 0.5 microg/g body weight (BW) of the steroid did not alter enzyme activity, and the higher dosages (1.0 and 2.0 microg/g BW) inhibited it significantly. Under in vitro conditions, addition of ascorbate and fructose (0.5-100 mM) to the incubation medium influenced enzyme activity differentially. At concentrations 0.5 and 1.0 mM, both fructose and ascorbate were ineffective except for the inhibition of testicular beta-Glc activity in the 1.0 mM ascorbate group. At higher concentrations (10, 50, and 100 mM), ascorbate inhibited enzyme activity in a concentration-dependent manner. At 10 mM concentration of fructose, only testicular beta-Glc activity was inhibited, but at higher concentrations (50 and 100 mM), activities of both enzymes decreased uniformly in a concentration-dependent manner. The addition of glucose had no significant effect on the enzyme activity at any of the concentrations tested. The results suggest that the inhibitory effect of testosterone on enzyme activity may be mediated through androgen-dependent metabolites, such as fructose and ascorbate.     

Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl–papain–Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 °C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 °C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.     

酶解鹿茸血ACE抑制活性及其与抗氧化活性关系的研究

实验采用木瓜蛋白酶和Alcalase碱性蛋白酶水解鹿茸血,在不同的时间取样,分别测定血管紧张素转化酶(ACE)抑制率、二苯代苦味肼基自由基(DPPH.)清除率和羟自由基(.OH)清除率。结果表明Alcalase蛋白酶水解产物ACE抑制活性和DPPH.清除活性较强,其水解3h的产物ACE抑制率为90.63%,DPPH.清除率为14.40%;木瓜蛋白酶水解5h的产物.OH清除率最高,为96.7%。用SPSS10.0软件分析结果,发现ACE抑制率与DPPH.清除率具有显著相关性。     

Change in antioxidant enzymes activity and some morpho-physiological characteristics of strawberry under long-term salt stress

The effects of long term salinity on some morpho-physiological characteristics were studied in strawberry Kurdistan and Queen elisa cultivars. Vegetative and biochemical traits were measured in strawberry cultivars subjected to three levels of salinity including 0, 40 and 80 mM at 20, 40 and 60th days after NaCl addition. Results showed that in both cultivars the dry weight of plant organs decreased in response to NaCl, except of crown weight in cv. Kurdistan. Root to shoot ratio increased due to a greater reduction in above ground biomass under salinity. Strawberry cultivars tended to decrease their stomatal conductance, RWC, proline, soluble carbohydrates and proteins during the different evaluation periods. Compared to the 20th day, peroxidase activity decreased at 80 mM during 40 and 60 days in cv. Queen elisa. On the contrary, ascorbate peroxidase activity elevated until the 40th day and decreased afterwards, in addition application of 40 and 80 mM NaCl increased the ascorbate peroxidase activity of both studied cultivars. Catalase activity increased from 20th until 60th days in cv. Queen elisa, while showed increase in cv. Kurdistan until day 40 and then decreased again at day 60. Application of 40 and 80 mM NaCl resulted in an increase in peroxidase, ascorbate peroxidase and catalase activities of both cultivars. The Queen elisa cv. showed lower tolerance index (45.88%) compared with cv. Kurdistan (67.97%). Finally, higher salinity resistance of cv. Kurdistan is probably associated with its ability to maintain higher RWC and higher activity of antioxidant enzymes.     

An improved procedure for the recovery of pancreatic enzymes with enhanced activity

Summary Pancreatic enzymes were recovered with enhanced activity by a newly designed extraction process. The protease activity was increased to the extent of 65% by using MES buffer, 64mM and duodenum enzyme extracts containing enterokinase at autolysis stage. The decrease in amylase activity due presumably to proteolytic degradation was effectively prevented by treating the enzyme solution with calcium prior to the autolysis.     

Time course of antioxidant responses of Capsicum annuum subjected to a progressive magnesium deficiency

The effect of magnesium (Mg²⁺)‐deficiency on the antioxidant responses of Capsicum annuum was investigated over a 60‐day period under controlled conditions. This Mg²⁺‐deficiency aimed to mimic the physiological conditions that plants may experience in the field. At each harvest time, five different leaf‐levels (L2 to L6) were distinguished. L2 and L6 correspond to the second and sixth youngest leaves, respectively. The following parameters were determined: Mg²⁺, chlorophyll and protein contents, total and redox pools of ascorbate and glutathione, and the activities of superoxide dismutase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. Under Mg²⁺‐deficiency, leaf Mg²⁺ contents decreased over time in all leaf‐levels except in the second youngest leaves (L2), where they remained constant at about 0.25% (dry weight basis). Mg²⁺‐deficiency led to an increase in the antioxidant enzyme activities concomitant with an increase in the ascorbate and glutathione pools, whereas total chlorophyll and soluble protein contents decreased. The L2 leaves showed an increase in glutathione reductase activity and in the ascorbate redox state whereas no difference was observed for the other parameters. Superoxide dismutase activities increased in L5 leaves from day 15 and, afterwards, in L3 to L5 leaves, irrespective of Mg²⁺ content. At day 30, glutathione reductase activities increased in L2 to L4 leaves and dehydroascorbate reductase activities in L4 leaves. At day 45, we observed an increase in the ascorbate peroxidase activities in L3 to L5 leaves. At the same time, ascorbate and glutathione pools increased in intermediate leaves, whereas chlorophyll content decreased in L3 and L4 leaves, and protein content decreased in L4 leaves. Results suggest that pepper leaves enhance their defence capacities against oxidative stress by increasing ascorbate more than glutathione synthesis. However, cells showed higher regeneration rates for the glutathione redox state than for the ascorbate redox state.     

The stereospecific d-glucose transport activity of cholate extracts from human erythrocyte membranes

The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at ?70°C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high d-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90 000, 23% of the polypeptides of molecular weight around 55 000, 30% of the phospholipids and at least 6% of the stereospecific d-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4°C the activity of the extrac decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore.

Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion-exchange and phenyl-Sepharose chromatography. The protein has an apparent subunit molecular mass of 57-58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 degrees C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases. Using sinigrin as the substrate Km was determined as 0.41 mM, and the kcat as 36 s(-1). With glucotropaeolin the Km and kcat values were determined as 0.52 mM and 22.8 s(-1), respectively. The enzyme was stable upon storage at 4 degrees C for many months, but lost some activity upon freezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to beta-glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.     

Purification and characterization of angiotensin I converting enzyme inhibitory peptides from the rotifer, Brachionus rotundiformis

Angiotensin I converting enzyme (ACE) inhibitory peptide was isolated from the marine rotifer, Brachionus rotundiformis. ACE inhibitory peptides were separated from rotifer hydrolysate prepared by Alcalase, α-chymotrypsin, Neutrase, papain, and trypsin. The Alcalase hydrolysate had the highest ACE inhibitory activity compared to the other hydrolysates. The IC₅₀ value of Alcalase hydrolysate for ACE inhibitory activity was 0.63 mg/ml. We attempted to isolate ACE inhibitory peptides from Alcalase prepared rotifer hydrolysate using gel filtration on a Sephadex G-25 column and high performance liquid chromatography on an ODS column. The IC₅₀ value of purified ACE inhibitory peptide was 9.64 μM, and Lineweaver–Burk plots suggest that the peptide purified from rotifer protein acts as a competitive inhibitor against ACE. Amino acid sequence of the peptide was identified as Asp-Asp-Thr-Gly-His-Asp-Phe-Glu-Asp-Thr-Gly-Glu-Ala-Met, with a molecular weight 1538 Da. The results of this study suggest that peptides derived from rotifers may be beneficial as anti-hypertension compounds in functional foods resource.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.     

Immunological characterization of rapeseed myrosinase

A purified 75-kDa myrosinase and a crude rapeseed myrosinase fraction were used as antigens to produce mouse anti-myrosinase monoclonal antibodies. The 75-kDa myrosinase was also used to produce a polyclonal rabbit antiserum. The antiserum and one monoclonal antibody reacted with three distinct rapeseed polypeptides of 75, 70 and 65 kDa (M75, M70 and M65, respectively). A second set of monoclonal antibodies reacted exclusively with the 75-kDa form of myrosinase, and a third set showed specificity towards two components of 52 and 50 kDa (myrosinase-binding proteins, MBP52 and MBP50, respectively). MBP52 and MBP50 lack inherent myrosinase activity, but are nevertheless capable of mediating immunoprecipitation of myrosinase due to their interaction with myrosinase. Gel chromatography and glycerol gradient centrifugation experiments resolved two myrosinase-containing fractions. One of these had an approximate molecular mass of 140 kDa and consisted of disulfide-linked dimers of the 75-kDa myrosinase. The other fraction was heterogeneous in size with molecular masses ranging from 250 kDa to approximately 1 MDa. The high-molecular-mass fractions contained complexes consisting of disulfide-linked 70-kDa and 65-kDa myrosinases and non-covalently bound 52-kDa and 50-kDa myrosinase-binding proteins.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptide from enzymatic hydrolysates of Styela clava flesh tissue

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the Styela clava flesh tissue. Nine proteases (Protamex, Kojizyme, Neutrase, Flavourzyme, Alcalase, pepsin, trypsin, α-chymotrypsin and papain) were used, and their respective enzymatic hydrolysates and an aqueous extract were screened to evaluate their potential ACE inhibitory activity. Among all of the test samples, Protamex hydrolysate possessed the highest ACE inhibitory activity, and the Protamex hydrolysate of flesh tissue showed relatively higher ACE inhibitory activity compared with the Protamex hydrolysate of tunic tissue. We attempted to isolate ACE inhibitory peptide from the Protamex hydrolysate of S. clava flesh tissue using ultrafiltration, gel filtration on a Sephadex G-25 column and high performance liquid chromatography (HPLC) on an ODS column. The purified ACE inhibitory peptide exhibited an IC₅₀ value of 37.1 μM and was identified as non-competitive inhibitor of ACE. Amino acid sequence of the peptide was identified as Ala-His-Ile-Ile-Ile, with a molecular weight 565.3 Da. The results of this study suggested that the peptides derived from enzymes-assisted extracts of S. clava would be useful new antihypertension compounds in functional food resource.     

Molecular mechanism of the inhibitory effect of cobalt ion on thermolysin activity and the suppressive effect of calcium ion on the cobalt ion-dependent inactivation of thermolysin

Thermolysin activity in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA) and FA-l-leucyl-l-alanine amide (FALAA) was examined at various Co(2+) and Ca(2+) concentrations. It decreased to 28% with increasing [Co(2+)] up to 18 mM. The Co(2+)-dependent inactivation was in part suppressed by adding Ca(2+) ion up to 0.5 mM, but 33% of the activity remained to be inactivated even with a sufficient concentration of Ca(2+) (>0.5 mM). The Co(2+)-dependent inactivation was shown to be composed of Ca(2+)-sensitive and Ca(2+)-insensitive parts. In the latter part which is observed at [Ca(2+)] >0.5 mM, Co(2+) plays as a competitive inhibitor. On the other hand, the Co(2+)-dependent inactivation in the Ca(2+)-sensitive part observed at [Ca(2+)] <0.5 mM proceeds time-dependently following second-order kinetics, and the time-course is in good agreement with that of decrease in the thermolysin band due to autolysis in SDS-PAGE. This indicates that Co(2+) accelerates the autolysis. Here, we describe the co-regulation of thermolysin activity by Co(2+) and Ca(2+) ions and propose a molecular mechanism for the inhibition of thermolysin by Co(2+) and suppressive effect of Ca(2+) on the Co(2+)-dependent inhibition. Co(2+) ion inhibits thermolysin activity not only as a competitive inhibitor but also promoting the autolysis.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

Autolysis of mu- and m-calpain from bovine skeletal muscle

The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.     

Endogenous inhibitor of angiotensin converting enzyme in the rat heart

We have identified a substance in the rat heart which inhibits ACE. This substance was characterized to be a sulfhydryl (SH) protein, the SH moiety being essential for the inhibitory activity. The inhibitory activity disappeared when the extract was boiled, or the ultrafiltrate(mol.wt.less than 10,000) was used, or when the extract was pretreated with the SH-blocking agent 5,5'-dithiobis-(2-nitro benzoic acid) at 0.5mM or the SH oxidizing agent diamide at 1mM. This substance was fractionated with Thiopropyl Sepharose affinity chromatography, precipitation with 40% ammonium sulfate saturation and high performance liquid chromatography. The mode of inhibition was competitive. In the presence of 20 micrograms/ml of this substance, the contraction of rat aortic strips induced by 5 x 10(-8)M ANG I was inhibited by 60%. This endogenous inhibitor of ACE may modulate the activity of ACE in the heart, in response to alterations in the oxidation-reduction balance in the tissue.