Levels Of Aminopeptidase Activity Research Articles

Evaluation of technological properties and selection of wild lactic acid bacteria for starter culture development

Sixty-six lactic acid bacteria strains, isolated from traditional cheeses, were identified by MALDI-TOF technology, characterised through the evaluation of their enzymatic activities (acidifying and proteolytic capacities and carboxypeptidase, aminopeptidase, dipeptidase and esterase activities) and selected through a scoring system based on activity results in order to select wild strains of technological interest for cheese manufacturing. The strains were identified as Lactococcus lactis (9), Leuconostoc citreum (3), Leuconostoc mesenteroides (2), Leuconostoc pseudomesenteroides (1), Levilactobacillus brevis (7), Lactiplantibacillus plantarum (32), Lactiplantibacillus paraplantarum (7) and Lacticaseibacillus paracasei (5). In general, Lactococcus lactis strains showed the highest degree of acidifying activity, especially in the first hours of fermentation, and extracellular proteolytic activity. In contrast, intracellular activities, assayed from cell-free extracts, were higher in the lactobacilli strains. L. paracasei strains showed the highest level of aminopeptidase activity, while some L. plantarum strains obtained high values of dipeptidase activity. Carboxypeptidase activity was very low or undetectable in many strains, although in others the activity values were exceptionally high. Esterolytic activity was generally low, although L. paracasei strains showed higher activity on short-chain substrates. Finally, 11 strains were selected using the scoring system that could be used in the design of starter cultures and co-cultures.

Neutral aminopeptidase and dipeptidyl peptidase IV in the development of collagen II-induced arthritis

This study evaluated the hypothesis that neutral (APN) and dipeptidyl-IV (DPPIV) aminopeptidase activity levels would be critical for the susceptibility to arthritis in collagen-induced model (CIA). The macroscopic signs of arthritis in CIA rats were checked and peripheral blood, synovial fluid and synovial tissue from knee joint were withdrawn. Soluble (SF) and solubilized membrane-bound (MF) fractions from the synovial tissue and peripheral blood mononuclear cells (PBMCs) were obtained. APN and DPPIV activities were fluorometrically quantified. Severe swelling in both the entire hind paws was the minimum criterion to select CIA rats with arthritis. These arthritic rats had high APN in plasma, synovial fluid and SF of the synovial tissue, together with low APN and DPPIV in MF of PBMCs and hallmark histological changes in tibio-tarsal joint. CIA rats with no macroscopic signs of arthritis were diagnosed as resistant and they had low APN in MF of the synovial tissue, low DPPIV in SF of PBMCs and high DPPIV in plasma together with histological aspects of tibio-tarsal joint similar to healthy control rats. Data suggested that APN and DPPIV activity levels are related to the development of arthritis, being protective or inducer of the susceptibility. Understanding what is controlling the compartment-specific changes of these peptidases and looking at ways in which to manipulate their activities may lead to a better knowledge of the arthritic processes and novel treatments.

Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese

Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains). Lactococcus strains were those that showed the greatest degree of acidifying and proteolytic activity. The cell-free extracts (CFE) of L. paracasei exhibited the highest level of aminopeptidase activity. The highest level of caseinolytic activity was shown by the CFE of one strain of L. lactis. High values were also obtained with the CFE of Lactobacillus and of several Leuconostoc. The highest level of dipeptidase activity was found amongst the strains of L. lactis. Carboxypeptidase activity was generally very low or undetectable for the majority of strains. The greatest degree of esterolytic activity was detected for Enterococcus.

Effect of intraperitoneal and intravenous administration of cholecystokinin-8 and apolipoprotein AIV on intestinal lymphatic CCK-8 and apo AIV concentration

CCK and apolipoprotein AIV (apo AIV) are gastrointestinal satiety signals whose synthesis and secretion by the gut are stimulated by fat absorption. Intraperitoneally administered CCK-8 is more potent in suppressing food intake than a similar dose administered intravenously, but the reason for this disparity is unclear. In contrast, both intravenous and intraperitoneally administered apo AIV are equally as potent in inhibiting food intake. When we compared the lymphatic concentration of CCK-8 and apo AIV, we found that neither intraperitoneally nor intravenously administered CCK-8 or apo AIV altered lymphatic flow rate. Interestingly, intraperitoneal administration of CCK-8 produced a significantly higher lymphatic concentration at 15 min than did intravenous administration. Intraperitoneal injection of apo AIV also yielded a higher lymphatic concentration at 30 min than did intravenous administration. Intraperitoneal administration of CCK-8 and apo AIV also resulted in a much longer period of elevated CCK-8 and apo AIV peptide concentration in lymph than intravenous administration. Furthermore, enzymatic activity of dipeptidyl peptidase IV (DPPIV) and aminopeptidase was higher in plasma than in lymph during fasting, and so, satiation peptides, such as CCK-8 and apo AIV in the lymph, are protected from degradation by the significantly lower DPPIV and aminopeptidase activity levels in lymph than in plasma. Therefore, the higher potency of intraperitoneally administered CCK-8 compared with intravenously administered CCK-8 in inhibiting food intake may be explained by both its higher concentration in lymph and the prolonged duration of its presence in the lamina propria.

Protease and esterase activity of staphylococci

The aim of this work was to characterize protease and esterase activities of staphylococci in order to establish if they could contribute to the release of amino acids and short-chain fatty acids during ripening of fermented sausages. Eighteen Staphylococcus strains belonging to the species Staphylococcus xylosus (5), S. saprophyticus (3), S. equorum (4), S. carnosus (4) and S. simulans (2), previously isolated from different types of Southern Italian fermented sausages, were screened for proteinase, aminopeptidase and esterase activities. Most of the staphylococci strains lacked detectable levels of proteinase activity against casein-fluorescein isothiocynate. In the active strains, this activity was extracellular or cell-envelope associated. The studied staphylococci strains also showed low levels of aminopeptidase activities, which preferentially hydrolysed substrates containing l-methionine, l-leucine and l-phenylalanine as N-terminal residue. In contrast, all staphylococcal strains possessed significant activity against short-chain fatty acid esters. The maximum esterase activities were detected in whole-cell suspensions and cell-free extracts and to a lesser extent in the extracellular medium. The substrates preferentially hydrolysed were ρ-nitrophenyl (ρ-NP) butyrate and ρ-NP caprylate and, secondly, ρ-NP palmitate. The extracellular extracts of most of the strains were only active against ρ-NP butyrate except for those of S. equorum (SI3, SI4) and S. simulans (Ssm12, Ssm21), which also hydrolysed ρ-NP caprylate and ρ-NP palmitate. The cell-free extracts and whole cells were mainly active against ρ-NP butyrate and ρ-NP caprylate, showing activity levels from 1760 U/mg of proteins to 54 U/mg of proteins and from 12,200 U/mg of proteins to 133 U/mg of proteins respectively. These activities were especially high in the strains that belonged to S. xylosus and S. equorum species. The diversity of the studied metabolic properties and, especially, the esterase activities in different staphylococcal species and even strains of the same species emphasize the relevance of these in vitro characterization studies for a rational selection of new starter cultures.

Molecular Characterization of Aminopeptidase N Cloned from Beet Armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)

Partial cDNA fragments of four homologous genes of aminopeptidase N (APN), known as a putative receptor of Bacillus thuringiensis (Bt) Cry toxin, were identified from Spodoptera exigua midgut using degenerate primers designed from conserved sequence of APN. Among them, one was determined to be predominant, and its full-length cDNA fragment (named as SeAPN) was cloned and characterized. The SeAPN encoding a 1, 007-amino acid protein possessed typical features of APN including signal peptide sequence, potential N-linked glycosylation sites, a zinc-binding metalloprotease consensus motif and GPI-anchor addition sequence. Sequence alignment showed high sequence similarity with other lepidopteran APNs known to be Cry toxin receptor. A full-length SeAPN construct was cloned into pIB/V5-His vector and transfected to Drosophila S2 cells. Enzyme preparation from the S2 cells expressing SeAPN exhibited a substantially high level of aminopeptidase activity, demonstrating its function as APN. Activated Cry toxin cocktail was prepared from B. thuringiensis subsp. aizawai HD-133, which known to be toxic against S. exigua, and used for the cytotoxicity test against the S2 cells expressing SeAPN. However, no symptom of cytotoxicity by the Cry toxin cocktail was observed.

Effects of morphine administration and its withdrawal on rat brain aminopeptidase activities

The endogenous opioid neuropeptide system seems to be involved in the neural processes which underlie drug addiction. Several studies have reported that the administration of morphine induces changes in the levels and/or activity of endogenous opioid peptides (enkephalin, dynorphin) and their precursors in specific brain regions of the adult CNS. The aim of this work was to study the effects of chronic morphine exposure and its withdrawal on certain aminopeptidases capable of degrading opioid peptides in brain areas including the amygdala, hypothalamus, hippocampus, striatum and brain cortices. In animals treated with morphine, aminopeptidase N presented higher enzyme activity levels in the striatum, the hypothalamus and the amygdala compared to control animals, although statistically significant differences were observed only in the case of the striatum. In addition, the activity of soluble puromycin-sensitive aminopeptidase (PSA) was found to be higher in the frontal cortex of these rats. In contrast, rats experiencing withdrawal symptoms presented decreased levels of aminopeptidase activity in certain brain areas. Thus, the activity of aminopeptidase N in the hippocampus and soluble puromycin-sensitive aminopeptidase in the frontal cortex were found to be lower in rats experiencing naloxone precipitated withdrawal symptoms, compared to the corresponding controls. Finally, the activity of the three studied aminopeptidases in vitro was unaltered by incubation with morphine, suggesting that the observed effects are not due to a direct action of this opioid upon the aminopeptidases. The results of the present report indicate that aminopeptidases may play an important role in the processes of tolerance and withdrawal associated with morphine administration.

GRP94-associated Enzymatic Activities: RESOLUTION BY CHROMATOGRAPHIC FRACTIONATION

GRP94 (gp96), which performs established functions as a molecular chaperone and immune system modulator, has been reported to display a number of intrinsic enzymatic activities, including ATP hydrolysis, protein phosphorylation, and aminopeptidase. In observing that GRP94 co-purified with bacterial beta-galactosidase through multiple chromatographic steps, we have examined the hypothesis that the reported enzymatic activities of GRP94 may reflect co-purification of contaminant enzymes, rather than intrinsic catalytic functions. In subjecting GRP94 to increasingly stringent chromatographic purification, we report that a GRP94 carboxyl-terminal directed protein kinase activity could be separated from GRP94 by heparin affinity chromatography. Analysis of the kinase substrate specificity indicates that this kinase is distinct from casein kinase II, which is known to co-purify with GRP94. Electrophoretically pure GRP94 displayed low, but significant levels of aminopeptidase activity. Further purification of GRP94 by anion exchange and heparin affinity chromatography yielded resolution of GRP94 from the aminopeptidase activity. Furthermore, exhaustive trypsinolysis of GRP94 preparations displaying aminopeptidase activity yielded complete proteolysis of GRP94 but did not affect aminopeptidase activity. These results are discussed with respect to current models for GRP94 function and the role of such co-purifying (poly)peptides in the generation of GRP94-dependent cellular immune responses.

Glucose Transporter Glut3 Is Targeted to Secretory Vesicles in Neurons and PC12 Cells

In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.

Physiological attributes of the pyloric caeca and anterior intestine of green sunfish ( Lepomis cyanellus) potentially influencing microhabitat specificity of Leptorhynchoides thecatus (Acanthocephala)

Pyloric caeca are present in some species of fish and serve as a habitat for many helminth parasites. Physiological and biochemical attributes of luminal contents of the caeca and proximal intestine of green sunfish, Lepomis cyanellus, were compared to determine factors that could account for localization of Leptorhynchoides thecatus (Acanthocephala) in caeca after initial establishment in caeca and anterior portion of the intestine. Caecal protein concentrations were significantly higher. The anterior intestine exhibited significantly higher levels of proteolytic activity; however, caeca exhibited higher levels of aminopeptidase activity. The pH of caeca was significantly higher than that of the proximal portion of the intestine. Caeca contained greater total amounts of materials, and therefore contained greater total amounts of proteins, free amino acids, lipids, carbohydrates, and bile salts. A significant time–place interaction was detected regarding total free amino acids, suggesting that enzymatic activity in the caeca was responsible for increased amounts of intestinal amino acids. Physiological factors that might influence microhabitat specificity of L. thecatus include inadequate levels of intestinal protein, different levels of proteolytic activity (perhaps resulting from differences in pH), such as aminopeptidase activity, and lack of sufficient quantities of anterior intestinal nutrients to sustain worms. These results demonstrate subtle spatial and temporal complexities intrinsic to the environment of helminths inhabiting the teleost enteric system.

Activities of aminopeptidases in a rat saline model of volume hypertension.

Aminopeptidase activity plays a role in the metabolism of several peptides that could be involved in blood pressure control. This activity has been implicated in the pathogenesis of hypertension, essentially in spontaneously hypertensive rats. However, few studies have examined aminopeptidase activities in animal models other than genetic hypertension. To analyze the aminopeptidase response to the specific conditions of the reduced renal mass saline model of arterial hypertension, aminopeptidase A activity (glutamyl- and aspartyl-aminopeptidase), aminopeptidase M activity (alanyl-aminopeptidase), aminopeptidase B activity (arginyl-aminopeptidase), pyroglutamyl-aminopeptidase and cystinyl-aminopeptidase were measured in the neurohypophysis, in the adrenal gland and in serum of this model of hypertension. In the neurohypophysis, there was a significant increase of soluble cystinyl-, alanyl-, arginyl-, pyroglutamyl- and membrane-bound aspartyl-aminopeptidase activities in hypertensive animals. In the adrenal gland, soluble cystinyl-, alanyl-, arginyl- and pyroglutamyl-aminopeptidase activities were also higher in hypertensive rats than in normotensive controls. No differences were found in serum levels of aminopeptidase activities between both groups of animals. A highly significant positive correlation between the neurohypophysis and the adrenal gland was observed for soluble cystinyl- and alanyl-aminopeptidase activities in the model of hypertension, whereas no correlation was observed in normotensive rats. Our results suggest that aminopeptidase activities could be involved in the regulatory response to the reduced renal mass hypertension and also suggest a coordinate response between the adrenal gland and the neurohypophysis, to the specific metabolic conditions of this model of hypertension.

Identification and characterization of proteolytic activity of Enterococcus spp. isolated from milk and Roncal and Idiazábal cheese

Roncal and Idiazábal cheeses are manufactured from ewe's milk in the Autonomous Region of Navarre in Spain. Levels of enterococci are high in these cheeses and in other types of ewe's-milk cheeses. The present study has identified enterococci present in the milk used and in the Roncal and Idiazábal cheeses after 120 days of ripening. A total of 282 strains were isolated and identified, and the cytoplasmic and extracellular enzymatic activities of some of the strains were assessed. The dominating species were Enterococcus faecalis, which accounted for 85% of the total both in the milk as well as in the two types of cheese, and Enterococcus faecium, Enterococcus durans, and Enterococcus avium which were present in lower proportions. Aminopeptidase and proteinase activity levels in enterococci were low, and considerable variation between strains of the same species was recorded, highlighting the need to study individual strains when selecting the most suitable bacteria as a starter culture. Aminopeptidase activity levels for the enterococci were appreciably higher at pH 7 than at pH 5.5, hence aminopeptidase activity by enterococci would appear to be less significant in the normal manufacturing conditions of the two cheeses.

Membrane-bound tyrosine aminopeptidase activities in the rat brain throughout the estrous cycle

Puromycin sensitive and insensitive membrane-bound aminopeptidase activity levels during the estrous cycle in several brain areas have been described in this research. We have found the highest aminopeptidase M activity levels during the proetrous stage in the hypothalamus, the amygdala and the pituitary gland. Since this enzyme has been involved in opioid peptide metabolism, it is suggested that aminopeptidase M could play a part in the decrease in the inhibitory influence of the endogenous opioids peptides that participate in the LH surge.

Determination of aminopeptidase activity in cheese

Aminopeptidase activity was studied in different types of cheese. Of the cheeses analysed having a mesophilous lactic flora, Roncal was contained the most aminopeptidase activity, coinciding with the highest ripening indices found [non-protein nitrogen (NPN) and amino nitrogen (N. NH2) with reference to the percentage total nitrogen (% TN)]. Of the Italian and Swiss type cheeses studied, the highest levels of aminopeptidase activity and NPN ripening index (% TN) were found in the Appenzeller and Parmiggiano-Reggiano cheeses. Among the mould-ripened cheeses, the blue cheeses had higher levels of enzyme activity and ripening indices [non-casein nitrogen (NCN), NPN and N. NH2 (%TN)] than the surface-flora cheeses. No changes in aminopeptidase activity of any importance were found in semi-hard ewes' milk cheese during ripening, over the first 3 months, but there was a considerable increase after 6 months of ripening.

Regional distribution of soluble and membrane-bound aminopeptidase activities in rat brain

Soluble and membrane-bound aminopeptidase activities in 11 regions of the rat brain were assayed using L-Leucine-2-naphthylamide as a substrate. In addition, two metabolic enzymatic activities were compared: lactate dehydrogenase and aspartate aminotransferase. All enzymatic activities showed significant regional differences when the data were analyzed statistically. Soluble aminopeptidase and aspartate aminotransferase activities were significantly lower in cortical than in subcortical areas. Membrane-bound aminopeptidase activity levels were higher in cortical areas. Lactate dehydrogenase activities did not differ between cortical areas and the rest of the zones studied. However, although no wide regional differences were found for the other enzymatic activities, membrane-bound aminopeptidase varied markedly across brain regions: a fivefold difference was observed between zones such as parietotemporal cortex and medulla. The differential distribution of this enzymatic activity is consistent with the hypothesis that it could be responsible for the enzymatic inactivation of some neuroactive peptides.

Neutral aminopeptidase activity levels during the estrous cycle and the pregnancy in the hypothalamus and the pituitary of the rat

Neutral aminopeptidase activity levels during the estrous cycle and pregnancy, in the hypothalamus and the pituitary of experimental animals, was studied in this research. The cyclic stages were the estrous, diestrous and proestrous (at morning and afternoon). The pregnancy phases were the 2nd, 7th, 14th and 20th day postinsemination. The parturition day was also studied. The higher cyclic enzyme levels were found in the proestrous stage (afternoon). No differences between estrous and diestrous were observed. During pregnancy, there was a rise at 7th and 14th day, with a decrease in the 20th day. In the hypothalamus, significant increases after parturition were shown, the latter not being observed in the pituitary. In general, the changes seem to be parallel to those of the luteinizing hormone.

Aminopeptidase activity levels during axonal and dendritic growth in the rat brain.

Brain aminopeptidase activity has been suggested as a candidate for the regulation and biotransformation of several neuropeptides. In this paper, changes in Lys- and Leu-aminopeptidase activities in rat brain hemispheres, cerebellum and medulla were examined in 1-, 3-, 5- and 7-days postnatal subjects. Aminopeptidase activities were studied by measuring the rate of hydrolysis of the artificial chromogenic substrates Lys- and Leu-2-naphthylamides (fluorimetrically detected in triplicate). Both enzyme activities show a decrease on the 3rd day of life followed by increases on the 5th and 7th day postbirth. It is suggested that these activities could play a part in the neurochemical changes that take place during axonal and dendritic growth in the rat brain.

The influence of type I collagen on the growth and differentiation of the human colonic adenocarcinoma cell line HT-29 in vitro

Abstract HT-29 Human colonic adenocarcinoma cells when grown on a plastic substratum were anaplastic in appearance and failed to express any morphological or biochemical features that were characteristic of intestinal differentiation. Growth of HT-29 cells subcutaneously in the flank of immune deprived mice gave rise to morphologically heterogeneous tumors which were poorly differentiated but contained approximately 11% of cells with an intestinal phenotype: these showed features typical of cell polarization with well-developed microvilli, tight junctional complexes and desmosomes between adjacent cells. The transfer of cells from plastic onto either a fixed (designated ‘non-released’) or floating (designated ‘released’) type I collagen gel induced some morphological features typical of intestinal differentiation; for example goblet-like cells were observed after 9 days, but biochemical markers of differentiation were expressed only modestly. The continued subculture of HT-29 cells on collagen type I gels, which were either attached to the plastic or floating in the medium, induced some morphological features of intestinal differentiation and changes in the activity of brush border-associated enzymes. Alkaline phosphatase activity was enhanced from 1.3×10 -3 μmoles/mg/min for cells cultured on plastic substrata to 2.1 × 10 -3 μmoles/mg/min when gels were non-released, and 2.9 × 10 -3 μmoles/mg/min when gels were released after 12 days of culture. This was confirmed by electron microscopical visualization of alkaline phosphatase activity. Elevated levels of aminopeptidase activity were also observed on day 12 (plastic = 26 milliunits/mg; non-released gel=41 milliunits/mg; released gel = 36 milliunits/mg). Similarly, changes occurred in the secretion of carcinoembryonic antigen from 0.96 × 10 -2 μg/mg/48 hours by cells cultured on plastic to 2.3 × 10 -2 μg/mg/48 hours by cells cultured on floating collagen gels. The effects of permitting HT-29 cells to undergo polarization were tested by culture on inert filter inserts: morphological features of intestinal differentiation were observed although this did not occur until after 21 days. These studies show that optimization of the growth conditions of anaplastic cells in vitro may provide cultures more representative of the tumor in vivo. This model system may be useful for cell biological and pharmacological studies of colon carcinoma.

Degradation of the neuropeptide proctolin by membrane bound proteases of the hindgut and ovary of Locusta migratoria and the effects of different inhibitors

AbstractProctolin was incubated in vitro with crude membrane preparations derived from hind gut and ovary of Locusta migratoria. The metabolites of proctolin were separated and identified by reversed phase‐high performance liquid chromatography which allowed the determination of the time course of degradation. At pHs 9.2 and 7.4, aminopeptidase activity predominated but some evidence of a carboxypeptidase could be inferred. At pH 6, aminopeptidase activity was slightly reduced compared to the level of aminopeptidase activity at the two former pHs; by contrast the carboxypeptidase was more important. At this pH, the presence of a membrane bound endopeptidase was detected.Phenanthroline, actinonin, amastatine, bestatin, and (2S,3R)‐3‐amino‐2‐hydroxy‐4‐(4‐nitrophenyl)‐butanoyl‐L‐leucine (HNBL) were tested at the concentration of 0.1 mM at pHs 8.2 and 6 and their ability to prevent the degradation of proctolin was estimated. A both pHs, HNBL and amastatin were revealed to be the most efficient inhibitors (50 to 75% inhibition) and o‐phenanthroline appeared particularly efficacious at pH 6 (70% inhibition), whereas it was relatively poor inhibitor at pH 8.2. Furthermore, the use of these inhibitors confirmed the importance of carboxypeptidase activity at pH 6.These data are the first to describe proctolin degradation in ovary and hindgut muscle, important targets of proctolin action.

Lodgepole pine seed germination. I. Changes in peptidase activity in the megagametophyte and embryonic axis

Lodgepole pine (Pinus contorta Dougl.) germination, as indicated by radicle emergence from the seed coat, was completed 2 days after imbibition at 28 °C. By this time, the hydrolysis of the megagametophyte storage protein reserve had commenced. This was 90% complete 5 days after imbibition. Megagametophytes and embryonic axes contained two classes of peptidases: aminopeptidases that hydrolyzed amino acid β-naphthylamides, and carboxypeptidases. Cell-free extract studies showed that carboxypeptidase activity was very low in mature seed megagametophytes and embryonic axes, but increased rapidly in these tissues after radicle emergence. However, maximum rates of carboxypeptidase activity only occurred when 70% of the megagametophyte storage protein reserve had been depleted 5 days after imbibition. Thus, it was unlikely that carboxypeptidases played a significant role in the rapid mobilization of megagametophyte storage proteins. Mature seed megagametophytes contained high levels of aminopeptidase activity. Following germination this level of activity was maintained in megagametophytes and only declined when storage protein hydrolysis was almost completed, 5 days after imbibition. It is proposed that aminopeptidases are involved in the hydrolysis of megagametophyte storage proteins in the germinated lodgepole pine seed.