In vitro digestibility of individual amino acids in rumen-undegraded protein: The modified three-step procedure and the immobilized digestive enzyme assay

Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy^II incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA value of the RUR. In conclusion, the modified TSP and IDEA kits are good approaches for estimating RUP-AA digestibility in soybean meal products, distillers dried grains with solubles, and fish meal samples.     

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.     

Pectin hydrolysis in a free enzyme membrane reactor: An approach to the wine and juice clarification

In this paper, a long-term investigation of the enzyme hydrolysis of pectin, using a free enzyme membrane reactor (FEMR), is presented. Production of crude enzymes (polygalacturonase and pectin lyase), in Aspergillus niger CECT 2088 cultures grown on an agricultural subproduct (apple pomace) as a low cost inducer, was also studied. Observing the retention of induced pectinases by the ultrafiltration membranes, the best FEMR configuration was achieved with the membrane of 10,000 nominal molecular weight limit (NMWL), since the biocatalyst was retained with no loss of enzyme activity. The monitoring of pectin hydrolysis in the FEMR was carried out by determining the viscosity and amount of reducing groups in both reaction mixture and permeate. A viscosity reduction of 88%, below the initial value, was reached in the reaction mixture only after 0.25 h of operation, while a gradual release of reduced sugars, in both reaction mixture and permeate, was detected. The FEMR was maintained in operation for 15 days, achieving an excellent catalytic efficiency.     

Creatin(in)e and Maillard reaction products as precursors of mutagenic compounds: Effects of various amino acids

The participation of creatin(in)e and Maillard reaction products in developing mutagenic activity was studied in model systems. Glucose and an amino acid were boiled under reflux for 2 h at 130°C together with creatine or creatinine dissolved in water-diethylene glycol (1:6). Threonine produced the highest mutagenic activity, 1068 revertants per μmol amino acid, towards TA98 after S9 activation, followed by glycine (410 rev/μmol) and lysine (246 rev/μmol). Proline, glutamic acid and the sulfur-containing amino acids produced less than 40 rev/μmol. Protein-bound amino acids produced no detectable mutagenic activity. When added to the reaction mixtures, the pure Maillard reaction products increased the mutagenic activity significantly. All precursors used occur in free form in meat. Work is now in progress to identify the mutagenic compounds produced in the model systems and to establish whether they also occur in fried meat.     

HPLC-fluorimetric method for analysis of amino acids in products of the hive (honey and bee-pollen)

An optimization of the OPA method has made feasible separation and quantification of 23 amino acids, which include 5 infrequently searched for. Detection limits ranged from 0.24 to 10.1 pmol in honey and from 29.1 to 0.42 pmol in bee-pollen; reproducibility (C.V.) ranged from 5.3% to 20.4%; recoveries were above 78.8%. Forty monovarietal honey samples from ilex, oak, heather and chestnut-tree were analyzed for their free amino acid profiles. α-Aminoadipic acid and homoserine are reported for the first time in honeys. Thirty-two samples of Spanish bee-pollen, made of a majority of pellets from Cistus Ladanifer (67.1%) and Echium plantagineum (8.9%), were analyzed for their free and total amino acid profiles. Free γ-aminobutyric acid was extensively found with an average of 0.53 mg/g, while Hser and Orn were infrequent. Manually separated monofloral pellets from Cistus ladanifer and Echium plantagineum were analyzed for their free amino acid contents (including proline): 32.46 and 21.87 mg/g for the former and 22.18 and 12.23 mg/g for the latter. In contrast, the total amino acid percentage (on a dry weight basis) was 13.95% for Cistus ladanifer and 32.22% for Echium plantagineum.     

Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus)

Mackerel (Scomber austriasicus) hydrolysates were prepared by an autolytic process and accelerated hydrolysis with a commercial enzyme, Protease N. Changes in the levels and compositions of free amino acids and small peptides during hydrolysis were investigated to find out their relationships with antioxidant activities. Increased levels of free amino acids, anserine, carnosine and other peptides of the hydrolysates obtained with protease were much higher than those by autolysis. Different antioxidant measurements including the inhibition of linoleic acid autoxidation, the scavenging effect on ,-diphenyl-β-picrylhydrazyl free radical, and the reducing power showed that mackerel hydrolysates possessed noticeable antioxidant activities. A good correlation existed between the amount of peptides and antioxidant activity. Three peptide fractions were separated from the hydrolysate by size exclusion chromatography. Results revealed that the peptide with molecular weight of approximately 1400 Da possessed a stronger in vitro antioxidant activity than that of the 900 and 200 Da peptides.     

Seasonal changes and preliminary characterization of cathepsin D-like activity in sardine (Sardina pilchardus) muscle

The seasonal changes of cathepsin D-like activity in white and dark muscle of sardine were followed and maximum activity was found in April and October, which could be related to the spawning period of this species. Higher catheptic activity was measured in dark muscle and females. The crude extract of this muscle enzyme had an optimum pH and temperature of 3.2 and 55 °C, respectively, and the activity was strongly inhibited by low levels of NaCl (6%). The thermal stability was lower at pH 3.2 than at pH 6.5 and the enzyme was stable in the pH range 3–4.5. The activity of this protease was detected during the ripening process of salted sardine which suggests its participation in this process.     

Primary metabolites and organic acid metabolism in apple (Malus sylvestris) fruit callus culture

In-vitro culture cells were obtained from seven different varieties of Indian apples (Malus sylvestris L). The cultivar Golden Delicious showed the highest yield of callus tissue followed by Maharaji and American Epirouge. Cultured apple cells exhibited some deviation from the apple fruit in primary metabolism as well as primary metabolite profiles. In callus cultures, the pool size of free amino acids and organic acids increased considerably while the free sugar pool decreased drastically compared with apple fiuit. There was higher incorporation of ¹⁴C acetate, ¹⁴C citrate, ¹⁴C malate and ¹⁴C succinate into the CO₂, lipid, protein, carbohydrate and amino acid fractions and lower incorporation into the free sugar fraction in cultured cells compared with the explant. The incorporation of ¹⁴CO₂ showed a similar trend. Qualitatively, there was some similarity between the callus and explant in free amino acid and sugar profiles and dissimilarity in organic acids. Compounds such as citrate, succinate and fumarate and also some amino acids (methionine, arginine, leucine and proline) were present at higher concentration in callus cultures whereas they were almost absent in the original tissue. There were also differences in the carbohydrate and protein profiles of explant ana callus as judged by their sugar and amino acid make-up respectively.     

Changes in composition of lipids,free amino acids and organic acids in rice-bran-fermented- sardine (Etrumeus teres) during processing and subsequent storage

Changes in composition of lipids, free amino acids and organic acids in rice-bran-fermented sardine—one of the traditional marine products of Japan—were investigated. The moisture content decreased rapidly during pre-curing and then slowly during rice bran fermentation. The salt content of the finished product was about 234 g kg⁻¹ on a dry weight basis. The pH decreased from 6.1 in the raw sardine to 5.3 in the finished product. Total volatile basic nitrogen increased up to 1.0 g kg⁻¹ after 2 months of rice bran fermentation and then remained almost unchanged. Most free amino acids except histidine and taurine increased during rice bran fermentation. The histamine content increased to 0.75 g kg⁻¹ at 2 months of rice bran fermentation and subsequently decreased gradually. Certain polyamines also accumulated on relatively lower levels. Lactic acid (8.14 g kg⁻¹ at 2 months of rice bran fermentation) was a prominent organic acid produced during processing. Considerable decompositions of triglyceride and phospholipids occurred accompanied by production and accumulation of a correspondingly high concentration of free fatty acids. The peroxide value and the amount of thiobarbituric acid reactive substances in the extracted total lipids decreased gradually during rice bran fermentation. It was concluded that lipid oxidation was restricted during rice bran fermentation although remarkable proteolysis occurred. The present traditional manufacturing process seems to be applicable to the technology of processing and subsequent preservation of fish products in the developing countries.     

Ruminal nitrogen metabolism and the passage of amino acids to the duodenum in sheep receiving diets containing hay and concentrates in various proportions

Three sheep fitted with rumen cannulas and three sheep fitted with duodenal re-entrant cannulas in addition to rumen cannulas were given, in successive periods, 700 g d^?1 of five diets consisting of chopped hay and a concentrate mixture of rolled barley and flaked maize (50:50) in the ratios of 1:0, 5:2, 3:4, 1:6, and 0:1. For bacteria isolated from the rumen of sheep fitted with rumen cannulas only, there were no significant differences in either the content of nitrogen in their dry matter or in the amino acid composition, but the content of α-?-diaminopimelic acid varied significantly (P < 0.05) between diets, mean values ranging from 39 to 50 kg^?1 nitrogen. As the proportion of concentrate in the diet increased, the intake of nitrogen was progressively reduced from 11.9 to 9.8 g d^?1 and this was accompanied by an increase in the amount of organic matter apparently digested in the rumen, from 296 to 402 g d^?1. But there were no significant differences between diets in either the ruminal concentration of ammonia-nitrogen (mean values varied from 78 to 96 mg litre^?1) or in the duodenal passage of total nitrogen, which ranged from 12.1 to 14.7 g d^?1 with a peak value for the 5:2 diet. Rates of synthesis of bacterial protein in the rumen were 186, 228, 124 and 94 g crude protein per kg organic matter apparently digested in the rumen for the 1:0, 5:2, 3:4, 1:6 and 0:1 diets respectively, the rates for the 1:6 and 0:1 diets being significantly (P < 0.05) lower than those for the other diets. The 0:1 diet was associated with a low proportion, 0.5, of bacterial protein in the duodenal crude protein and it is suggested that with this diet, protozoal protein made an appreciable contribution to the crude protein passing from the rumen.     

Kinetics of free protein amino acids, free non-protein amino acids and trigonelline in soybean (Glycine max L.) and lupin (Lupinus angustifolius L.) sprouts

A contribution for a better nutritional knowledge of lupin (Lupinus angustifolius L. var. zapaton) and soybean (Glycine max L. var. merit and var. jutro) sprouts about free protein amino acids (FPAA), free non-protein amino acids (FNPAA such as β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP), α-aminoadipic acid, γ-aminobutyric acid (GABA) and β-alanine) and trigonelline content has been carried out. Seeds were germinated at 20 °C and darkness during several days in order to obtain good appearance sprouts. The content of FPAA and FNPAA varied among species and varieties. FPAA increased dramatically during germination and the lowest concentrations were found for G. max L. var. jutro. Cys was not detected as FPAA in seeds and seedlings of both legumes. Asn was present in the highest amounts in soybean and lupin seedlings. The α-aminoadipic acid gliotoxin, which was not present in raw soybean seeds, appeared in low amounts during germination. Later stages of germination caused significant increases of β-alanine and GABA, amino acids with beneficial properties. Germination also affected the content of the multifunctional plant hormone trigonelline and slight changes were observed. Highest levels of FPAA, beneficial FNPAA and trigonelline were found at 4, 6 and 9 days of germination for G. max L. var. jutro, G. max L. var. merit and L. angustifolius L. var. zapaton, respectively. Furthermore, these selected germination conditions showed low levels of the gliotoxic α-aminoadipic acid ensuring nutritional safety of the studied seedlings.     

Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish (Feseekh) during ripening and storage

The aim of this study was to investigate changes in the formation of amino acids and biogenic amines in Egyptian salted-fermented fish (Feseekh) during ripening (20 days) and storage (40–60 days). The total concentration of free amino acids increased from 8 (dry weight; DW) to 72 g/kg (DW) after 60 days of storage. The predominant free amino acids were leucine, glutamic acid, lysine, alanine, valine, aspartic acid, isoleucine and citrulline. Their concentrations accounted for 68% of the total concentration of amino acids after 60 days. The total contents of biogenic amines ranged from 84 to 1633 mg/kg (DW) during the investigated period. Cadaverine was the major amine detected in Feseekh at all sampling stages and its concentration varied between 21 and 997 mg/kg (DW). The histamine content (211 mg/kg DW) only exceeded the maximum tolerance level (200 mg/kg) after 60 days. It could be concluded that Feseekh can be consumed without any health risks between 20 and 40 days but it can be hazardous after 60 days due to the biogenic amine content.     

Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicans GLY1 is AF009967.     

Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus B-6 grown on Avicel under aerobic conditions

A multienzyme complex, cellulosome, of the facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6 was produced on microcrystalline cellulose (Avicel) under aerobic conditions. During growth on Avicel, the bacterial cells were found to be capable of adhesion to Avicel by scanning electron microscopic (SEM) analysis. The multienzyme complex of P. curdlanolyticus B-6 was isolated from the crude enzyme preparation by gel filtration chromatography on Sephacryl S-300 and affinity purification on cellulose. The isolated multienzyme complex was able to bind to both Avicel and insoluble xylan and consists of cellulolytic and xylanolytic enzymes such as avicelase, carboxymethyl cellulase (CMCase), cellobiohydrolase, β-glucosidase, xylanase, β-xylosidase and α-l-arabinofuranosidase. The molecular mass of the complex was estimated to be 1600 kDa. It composed of at least 12 proteins on SDS-PAGE and 10 CMCases and 11 xylanases on zymograms. The isolated multienzyme complex could degrade the raw lignocellulosic substances effectively.     

Isolation and characterization of a hexokinase mutant of Schwanniomyces castellii: Consequences on cell production in continuous culture

A mutant of Schwanniomyces castellii with reduced glucose phosphorylation and with practically no phosphorylation of fructose was investigated. Carbon catabolite represion of α-glucosidase and amylases was reduced. Repression of β-galactosidase was normal. We have compared in continuous culture this mutant strain with wild type and another previously described mutant. The relationship between the specific rate of glucose consumption (Qs) and residual glucose concentration (s) in an inverse mode, suggests that there may be two types of transport of glucose. Mutation at the phosphorylation level causes apparent modification of the kinetic parameters of glucose uptake rate. The consequence of mutation at the phosphorylation level on biomass production was discussed.     

Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h⁻¹, whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h⁻¹. Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.     

The apparent ileal digestibility of amino acids and the digestible energy content of naked oats (Avena sativa cv Bandicoot) fed to growing pigs

An experiment was conducted to determine the apparent ileal digestibility of amino acids and the digestible energy (DE) content of two samples of naked oats (Avena sativa cv Bandicoot) and to compare these parameters in wheat (Triticum aestivum cv Machete) and dehulled oats (groats; Avena sativa cv Echidna). Four Large White male pigs were fitted with simple T-piece ileal cannulae and allocated to experimental diets in a 4×4 Latin square design. Amino acid digestibility coefficients were determined after continuous eight hour collections of digesta over two consecutive days using acid-insoluble ash as an indigestible marker. Digestible energy was determined using grab samples of faeces. No significant difference between the four test cereals was found in the digestibility of all amino acids, except for proline and lysine. The apparent ileal digestibility of lysine in wheat (0·87) and two samples of naked oats (0·89 and 0·82, respectively) was lower (P<0·05) than dehulled oats (0·91). The mean DE value of the naked oats samples was 16·96 MJ kg⁻¹ (air-dry basis). The results suggest that Bandicoot naked oats and dehulled oats are superior amino acid and DE sources to wheat and have potential for use in weaner and grower pig diets. © 1998 SCI.     

Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses

Hard cooked cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food. In this work, we assessed the proteolysis produced by enzymes of two strains of L. helveticus in a new cheese model, which consisted of a sterile substrate prepared with hard-cooked cheeses, and identified the time of ripening when main changes in proteolysis are produced. The extract, a representative model of the aqueous phase of the cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90, and 180 days) made with starters composed of the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 ??m). The substrates were incubated at 34 °C during 21 days; samples were taken at 0, 3, 7, 14, and 21 days. Sterility was verified by plating samples on skim milk agar and incubating at 37 °C for 48 h. Proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Great variation in peptide profiles was found as incubation progressed in cheese extracts, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the extracts. The extracts derived from cheeses with L. helveticus SF138 showed low production of peptides and a notable increase in free amino acids content during incubation. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, but the free amino acids accumulation was lower than in the first case, which suggested that L. helveticus SF209 had either a low peptydolitic activity or produced an intense amino acids breakdown. This trend was more evident for extracts prepared with 90-day-old cheeses. It was concluded that the strains of L. helveticus assayed showed potentially complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. The extract was an appropriate medium to model hard cooked cheese ripening in short periods of time.     

Isolation and characterization of branched chain fatty acids (other than those derived from phytol) in cod liver oil

A group of mono- and di-unsaturated branched chain fatty acids were isolated and characterized by GC-MS. A GC-MS analysis of the perhydroderivatives of these acids revealed the presence of the following acids: 5 - methyltetradecanoic, 5,7-dimethyltridecanoic, 7-methylhexadecanoic, 5,7- dime t h y 1 te tradecanoic, 7,9-dime t hyl hexadecanoic, 9-met h yloctadecanoic, 7,9- and 9,11-dimethyloctadecanoic acids. Of these, only the 7,9- dimethylhexadecanoic acid was fully characterized in its unhydrogenated form as 7,9-dimethyihexadecadienoate (Δ 63). The presence of the branched methyl groups on alternate odd carbon atoms is uncommon.     

Pepsinogen and pepsin from the stomach of smooth hound (Mustelus mustelus): Purification,characterization and amino acid terminal sequences

Pepsinogen from the stomach of smooth hound (Mustelus mustelus) was purified to homogeneity by 20–70% ammonium sulphate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose anion exchange chromatography with a 9.4-fold increase in specific activity and 38.36% recovery. Upon activation at pH 2.0, M. mustelus pepsinogen was converted to active form in one-step pathway. Molecular weights of the purified pepsinogen and the active pepsin were estimated to be 40,000 and 35,000 Da using SDS-PAGE and gel filtration, respectively. The optimum pH and temperature for the pepsin activity were pH 2.0 and 40 °C, respectively, using haemoglobin as a substrate. Activity was completely inhibited by Pepstatin A but not by phenylmethylsulphonyl fluoride, a serine-protease inhibitor and ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. The N-terminal amino acid sequences of the first 15 amino acids of the activation segment of the pepsinogen and the first 20 amino acids of the active pepsin were LLRVPLRKGKSTLDV and ATEPLSNYLDSSYFGDISIG, respectively. M. mustelus pepsinogen, which showed high homology to rat C pepsinogen, had Thr-Leu-Asp sequence at amino acid positions 12–14 not found in all pepsinogen sequences. A remarkable substitution was found in the activation segment of M. mustelus pepsinogen: the Arg-13 conserved in all gastric proteinases, whose sequences are known, is replaced by Leu-13.