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PubMed1973

Studies on the turnover of protein and glycoprotein components in rabbit kidney brush borders.

The kinetics of incorporation of [(3)H]lysine and [(14)C]glucosamine into kidney brush borders were studied in vivo. The patterns of incorporation and loss of radioactivity from the brush borders were similar for both radioactively labelled precursors. Maximal labelling occurred 15-20h after injection of the precursors, and then the radioactivity declined rapidly until 50h. The radioactivity of brush borders then remained constant until 120h and thereafter declined slowly. These results are interpreted as indicating that two processes contribute to the turnover of brush-border components, pinocytosis and a slower turnover of the components of the microvilli. Studies of the distribution of radioactivity among glycoprotein components of the brush borders, separated by polyacrylamide-gel electrophoresis, indicated that the membrane components turned over in unison.

Animals↗
PubMed1977

Influence of cations at the plasma membrane in controlling polysaccharide secretion from sycamore suspension cells.

1. Three soluble polysaccharides and a soluble protein containing hydroxyproline were secreted by sycamore suspension cultures. l-[1-(3)H]Fucose was incorporated solely into the fucose of fucoxyloglucan and l-[1-(14)C]arabinose mainly into the arabinose of arabino-galactan. [U-(14)C]Glucose was a general precursor for soluble protein and polysaccharides. 2. The steady-state rate of secretion of all the polymers was increased within seconds of adding various electrolytes and polyelectrolytes to the growth medium. The increased secretion was induced by cations at the outer surface of the plasma membrane. It was brought about by a stimulation of the normal mechanisms of cell-wall polysaccharide secretion. It was partly inhibited by anaerobiosis or sodium arsenate and was unaffected by temperature changes in the range 0-35 degrees C. 3. The precursor pool from which secretion was induced contained completely synthesized polysaccharides and was probably located in the Golgi-derived vesicles. The results indicated that the endoplasmic reticulum did not secrete polysaccharide directly to the cell exterior. 4. The various cations probably induced secretion by causing a depolarization of the negative electric potential of the cell surface, and this resulted in the fusion of vesicles with the plasma membrane. 5. Analogy with exocytosis and pinocytosis in various animal tissues suggested that the decreased surface potential brought about membrane fusion by causing an increase in plasma-membrane permeability to Ca(2+). 6. The results showed that the fusion of vesicles with the plasma membrane was rate-limiting and a potential control point. Auxin-stimulated cell-wall deposition could be a result of a stimulated influx of Ca(2+) causing vesicle fusion with the plasma membrane.

Calcium Chloride↗
PubMed1978

Uptake and degradation of iodine-labelled chylomicron remnant particles by monolayers of rat hepatocytes.

1. Rat chylomicrons were labelled with 125I with 69--72% of the iodine in the protein moiety. Less than 1 nmol of iodine was incorporated per nmol of protein. Of the peptide radioactivity 44--56% was in apolipoprotein A-1, 30--40% in the C peptides and 11--15% in apolipoprotine B. The arginine-rich peptide, which accounted for about 14% of the chylomicron protein mass as determined by scanning of sodium dodecyl sulphate-polyacrylamide gels, contained very little radioactivity. 2. Chylomicron remnants generated with postheparin plasma from iodine-labelled chylomicrons showed a relative increase in the percentage of the arginine-rich peptide (76--90% of the apolipoprotein mass according to gel scanning). The major portion of the peptide iodine label was present in apolipoprotein A-1 (43--57%), B (22--32%) and C peptides (17--35%). 3. When iodine-labelled chylomicron remnants were added to rat hepatocytes in primary culture, labelled peptides were taken up and degraded by the hepatocytes by a saturable process. The Vmax. for the uptake was calculated to the 300ng of protein/h per mg of cell protein and the apparent Km as 7.7 microgram of protein/mg of cell protein. A larger proportion of the 125I-labelled lipids of the remnants (mainly polar lipids) was taken up. This suggest that these may also enter the cells by a mechanism that does not involve particulate uptake, such as phospholipid exchange. 4. The degradation of labelled peptides was inhibited by colchicine, concanavalin A, chloroquine and NH4Cl, which also inhibit degradation of the cholesteryl ester portion. All these drugs exerted their inhibition mainly after the uptake of labelled peptide. No degradation occurred at 4 degrees C, and also the uptake was markedly decreased. 5. The uptake of labelled chylomicron remnant peptide was 77 times as effective as that of labelled sucrose, which is likely to be taken up randomly by pinocytosis.

Ammonium Chloride↗
PubMed1979

Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments.

1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.

Cells, Cultured↗
PubMed1979

Macrophage protein turnover. Evidence for lysosomal participation in basal proteolysis.

1. Turnover of intracellular proteins in cultured mouse macrophages was found to be slightly accelerated by the omission of serum from the culture medium. Media containing 10% (v/v) or more of serum established basal degradation rates in the cultures. 2. Basal degradation rates varied considerably between experiments, probably as a result of variable activation in vivo of the macrophages. 3. The selective carboxyl proteinase inhibitor pepstatin, which appeared to enter the lysosomes of the cells by pinocytosis, gave a progressive inhibition of basal proteolysis up to a maximum of about 40%. Cellular cathepsin D was largely inhibited after 48h of cultivation with pepstatin (100 micrograms/ml). 4. Leupeptin and 7-amino-1-chloro-3-tosylamidoheptan-2-one are less selective proteinase inhibitors. They also induced 25--35% inhibition of degradation, but their actions may not have been restricted to lysosomes. 5. Several solutes and particles that are endocytosed by macrophages and stored in lysosomes induce some inhibition of basal proteolysis, whether or not they themselves are substrates for proteolysis. 6. Colchicine was without effect on protein degradation, but cytochalasin B and the local anesthetics lidocaine and procaine, all of which have effects on microfilaments, were significantly inhibitory. This inhibition may result from a decrease in the rate of autophagy, and thus of lysosomal proteolysis, due to prevention of microfilament action.

Animals↗
PubMed1979

Copper metabolism in mottled mouse mutants: copper concentrations in tissues during development.

The copper content of various organs of ;brindled' female heterozygotes and male mice affected by this X-linked mutation are documented at the last day of intrauterine development, at 1 day after birth and at 11 days of age. The findings indicate defective placental transfer of copper in utero, and an even more marked defect in intestinal absorption of copper after birth. In addition there is an abnormal distribution of copper among the tissues of the body once it is absorbed. The mutation produces abnormal accumulation of copper in kidney, in gut mucosa and in testis, whereas liver, brain, plasma and most other organs show diminished copper concentrations. The intestinal malabsorption of copper is accompanied by accumulation of abnormal amounts of the metal in the intestinal-mucosa cells. Copper concentrations in both mucosa and luminal contents rise progressively from duodenum to ileum. Defective upper-intestinal absorption, consequent progressive increase in luminal copper concentration and pinocytosis in the ileum would seem to explain the findings. Radioisotopic studies eliminated the possibility of excessive excretion of copper in bile or across the intestinal mucosa. Detailed comparison with findings in humans with Menkes' syndrome is difficult because of the different stages of development at which the studies have been performed, but the results seem in general to conform very satisfactorily. Those differences seen are probably explicable by known species differences. All the findings are in accord with a hypothesis that the basic defect involves accumulation and retention of copper in the cells of affected tissues such as kidney, gut mucosa and placenta.

Animals↗
PubMed1979

Copper metabolism in mottled mouse mutants: distribution of 64Cu in brindled (Mobr) mice.

1. Duodenal injection of 64Cu in treated adult mutant mice (Mobr/y) revealed severe malabsorption of copper. In suckling mutants, malabsorption was less severe, owing to delayed absorption between 2 and 5 h after injection. Pinocytosis at the distal small intestine seems the likely explanation for this difference, and this is supported by results of ileal injection of radioisotope in the suckling mice. 2. The distribution of 64 Cu in various organs was measured in suckling normal, mutant and heterozygote mice and in adult normal and mutant mice during 48 h after intracardiac injection. Excessive accumulation of radioisotope was observed in most extrahepatic organs of mutant and heterozygote mice and was most pronounced in kidney. This could not be explained by initial copper deficiency. The livers of suckling mutant and heterozygote mice lost radioisotope rapidly after normal initial uptake. This pattern was not seen in adult mutants.

Animals↗
PubMed1980

The inhibition of macrophage protein turnover by a selective inhibitor of thiol proteinases.

1. A new inhibitor of thiol proteinases, benzyloxycarbonylphenylalanylalanine diazomethyl ketone (benzyloxycarbonylphenylalanylalanyldiazomethane, Z-Phe-Ala-CHN2) was added to cultured mouse peritoneal macrophages prelabelled with [14C]leucine. The degradation of protein was studied under conditions of basal proteolysis in the presence of 10% pig serum. After a lag of about 6 h a time- and dose-dependent inhibition of protein degradation was observed, up to a maximum of about 40%. 2. The inhibitor entered the cells with kinetics consistent with entry by pinocytosis, giving access to the lysosomal system. 3. Intracellular cathepsin B was almost completely inactivated after 90 min of exposure of the culture to 0.1 mm-inhibitor. 4. The inhibition of proteolysis and of cathepsin B was reversed virtually completely within 24 h, when the inhibitor was removed from the medium. Since the inhibitor forms a covalent bond with the enzyme, the recovery of cathepsin B activity presumably reflects production of new molecules of active enzyme. 5. The inhibitory effects of pepstatin, the carboxyl proteinase inhibitor, were under some circumstances additive with those Z-Phe-Ala-CHN2, and were also largely reversible. 6. It is concluded that thiol proteinases play a major role in lysosomal proteolysis in cultured macrophages.

Animals↗
PubMed1980

The role of extra-hepatic tissues in the receptor-mediated plasma clearance of glycoproteins terminated by mannose or N-acetylglucosamine.

The mannose- and N-acetylglucosamine-specific pathway for the clearance of mammalian glycoproteins has been characterized by using 125I-labelled neoglycoproteins, glycosidase-treated orosomucoid and lysosomal glycosidases (beta-glucuronidase and beta-N-acetylglucosaminidase) as probes. There are two components to this pathway in vivo; one liver-dependent and the other extrahepatic or liver-independent. Cells that mediate clearance by the latter component of the pathway are present in spleen, bone and in elements of the reticuloendothelial system, but not in the kidney. Glycoproteins that possess terminal mannose, glucose or N-acetylglucosamine residues, including various lysosomal enzymes, are rapidly cleared from plasma via this pathway. Glucose-terminated glycoproteins are recognized by two pathways in the intact animal; the hepatic galactose-specific pathway and the mannose/N-acetylglycosamine-specific pathway, which is present in liver and in peripheral tissues. Following removal of the liver by surgical evisceration, glucose-terminated glycoproteins are cleared whereas glycoproteins bearing galactose are not cleared. Uptake of 125I-labelled neoglycoproteins and agalacto-orosomucoid by isolated alveolar macrophages closely mimics clearance in vivo by the mannose/N-acetylglucosamine pathway. Neoglycoproteins terminated by mannose, glucose or N-acetylglucosamine all compete with 125I-labelled agalacto-orosomucoid for uptake by receptor-mediated pinocytosis. The extent of substitution of the neoglycoproteins is a critical determinant of their inhibitory potency. It is proposed that mononuclear phagocytes are in important component of the clearance pathway in vivo. The mannose/N-acetylglucosamine pathway may be important in the regulation of extracellular levels of various glycosylated macromolecules, including lysosomal hydrolases.

Abdomen↗
PubMed1982

A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

Animals↗
PubMed1982

Cystine accumulation in cystinotic fibroblasts from free and protein-linked cystine but not cysteine.

The accumulation of cystine in cystinotic fibroblasts from free and protein-linked cystine has been investigated. Cystine is not accumulated from cysteine but is readily accumulated from cystine. The accumulation from free cystine does not occur as a result of pinocytosis or from the degradation of a rapidly metabolized protein pool. Further studies of the degradation of disulphide-containing proteins by these cells may aid understanding of the mechanisms of proteolysis.

Cell Line↗
PubMed1988

The calcium ionophore A23187 increases the tight-junctional permeability in rat liver.

The effect of an increase in intracellular Ca2+ concentration on tight-junctional permeability in rat liver was studied by using the calcium ionophore A23187. Infusion of 100 microliters of dimethyl sulphoxide containing various amounts of A23187 over 30 min into isolated perfused livers was followed by a pulse of horseradish peroxidase (HRP) under single-pass conditions. The first biliary HRP peak, a measure of junctional permeability, was increased 4-fold with 100 micrograms of A23187. There were, however, no significant effects on bile flow or on aspartate aminotransferase leakage as compared with the control at this dosage, and thus the increase in junctional permeability was occurring without evidence of appreciable cholestatic or hepatocellular damage. Higher dosages of A23187, however, caused not only an increase in HRP peak height but also changes in bile flow and increases in aminotransferase leakage, indicating more extensive effects at these higher dosages. A second peak of HRP secretion, occurring 20-25 min after the HRP pulse, was also elevated approx. 3.5-fold; this may indicate that pinocytosis and transcellular movement of HRP are also increased under these conditions.

Animals↗
PubMed1992

Inhibition of cysteine proteinases in lysosomes and whole cells.

Inhibitors of cysteine proteinases have been used extensively to dissect the roles of these proteinases in cells. Surprisingly though, little work has been performed to demonstrate unequivocally that the inhibitors reach and inactivate their target proteinases in cell culture or in vivo. In the present study, the permeability of lysosomes and whole cells has been studied. Benzyloxycarbonyl (Z)-[125I]iodo-Tyr-Ala-diazomethane (CHN2), an inhibitor of cathepsins L and B, has been shown to label active forms of these enzymes in lysosomes and whole cells. The ability of other cysteine proteinase inhibitors to block this labelling has been used to indicate the permeation of these compounds. All the inhibitors were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomal extracts. In intact lysosomes or cells, however, only N-[N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-leucyl]-3- methylbutylamine ('E-64d') Z-Tyr-Ala-CHN2, Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2. N-[N-(L-3-trans-Carboxyoxirane-2-carbonyl)-L-leucyl]amino-4-gua nidinobutane (E-64) and leupeptin were unable to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomes or in cells. The ability to block labelling in lysosomes is an indication of the ability of the inhibitor to diffuse across membranes. Thus E-64 and leupeptin do not readily permeate membranes and therefore their uptake into cells probably only occurs via pinocytosis.

3T3 Cells↗
PubMed2000

Unconventional myosins.

Myosins constitute a large superfamily of F-actin-based motor proteins found in many organisms from yeast to humans. A phylogenetic comparison of their head sequences has allowed them to be grouped into 15 different classes. Unconventional myosins can be monomeric or dimeric, but are thought not to form filaments, unlike conventional myosin. The double-headed class-V myosins are good candidates for transporting vesicles, organelles and (mRNA) particles along actin filaments. Class-I myosins are involved in membrane dynamics and actin organization at the cell cortex, thus affecting cell migration, endocytosis, pinocytosis and phagocytosis. A class-III myosin from Drosophila is required for phototransduction and maintenance of the rhabdomere. Class-IX myosins negatively regulate the small G-protein Rho, a signalling molecule that regulates the organization of the actin cytoskeleton. Protein kinases that are regulated by members of the Rho small G-protein family regulate the motor activities of different myosins.

Animals↗
PubMed2000

Uptake of the yeast Malassezia furfur and its allergenic components by human immature CD1a+ dendritic cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with increasing prevalence, though still little is known of the pathomechanisms and the causes of the disease. Patients with AD often have specific IgE reactivity to the yeast Malassezia furfur (M. furfur), present in the normal microflora on human skin. To investigate the possible interaction of immature and mature antigen-presenting dendritic cells with the yeast M. furfur and its allergenic components. Monocyte-derived dendritic cells (MDDCs) generated from human peripheral blood were allowed to interact with FITC-labelled whole M. furfur yeast cells, M. furfur extract, a recombinant allergen from M. furfur designated rMal f 5 and M. furfur mannan, in the absence of IgE antibodies. Interaction and uptake were detected using flow cytometry and confocal laser scanning microscopy. Internalization of M. furfur yeast cells and yeast components by immature MDDCs was found using confocal laser scanning microscopy. Results from flow cytometric studies showed that a median of 94% (range, 65-98%) of the immature CD1a+ MDDCs were M. furfur extract positive, 81% (75-97%) rMal f 5 positive and 93% (62-98%) mannan positive. Mature CD1a+ MDDCs were significantly less efficient in this respect, with the corresponding figures only 26% (6-37%, P < 0.01), 6% (2-15%, P < 0.05) and 32% (9-50%, P < 0.01), respectively. Uptake of the non-glycosylated rMal f 5 by immature CD1a+ MDDCs was decreased to 27% (15-38%) by inhibition of pinocytosis. The binding of M. furfur extract and mannan was inhibited in a dose-dependent manner by methyl-alpha-D-mannopyranoside, suggesting uptake via the mannose receptor. Human immature CD1a+ MDDCs can efficiently take up M. furfur and allergenic components from the yeast in the absence of IgE antibodies, implying that sensitization of AD patients to M. furfur can be mediated by immature dendritic cells in the skin.

Allergens↗
PubMed1998

Cellular 'neurothekeoma': an epithelioid variant of dermatofibroma?

AIMS: Cellular neurothekeoma is a rare benign cutaneous neoplasm with conflicting opinions regarding its histogenetic origin (nerve sheath, smooth muscle, myofibroblasts) as well as its relation to myxoid neurothekeoma (nerve sheath myxoma). The present series describes 15 cases whose clinicopathological features indicate a relationship to dermatofibroma. METHODS AND RESULTS: In this retrospective clinicopathological study, the lesions preferentially occurred in adolescents to young adults on the upper half of the body, often clinically diagnosed as some kind of fibrohistiocytic tissue response. Besides characteristic whorled nests to fascicles of palely eosinophilic epithelioid cells, all lesions showed variable clues pointing to dermatofibroma: acanthosis, ill-defined storiform periphery, peripherally accentuated prominent sclerosis and lymphocytic demarcation/infiltration. Immunohistochemically, all cases were positive with NK1C3 (CD57), KiM1p and proliferating cell nuclear antigen, seven were positive for neurone specific enolase, five for factor XIIIa, six for smooth muscle specific actin and three for E9, an antimetallothionein marker. These findings are similar to those of conventional dermatofibromas, the variability of the profile being best explained by time cycle and function dependent changes. Ultrastructurally, two cases showed microfilaments, attachment plaques, prominent pinocytosis and focal remnants of basal lamina. A careful study of the data and photomicrographs from the literature reveals that in many cases similar conclusions could be reached. Obvious discrepancies are most likely due to the confusion with myxoid neurothekeoma, a well circumscribed, more spindly and myxoid, S100 positive lesion of Schwannian origin. CONCLUSION: The appearance of dermatofibromas is markedly influenced by architectural, e.g. in deep penetrating dermatofibroma, and/or cellular/stromal criteria, e.g. in epithelioid cell histiocytoma or sclerosing dermatofibroma. Cellular neurothekeoma seems to be a variant of dermatofibromas with both architectural and cellular/stromal peculiarities, i.e. plexiform pattern, epithelioid cytology and stromal sclerosis.

Actins↗
PubMed1996

Augmented antigen presentation by mouse Ia + T clone cells BK-BI-2.6.O4.1 mediated by transferrin receptors.

The murine T clone cells BK-BI-2.6.O4.1 (BI/O4.1) synthesize and express MHC class II molecules constitutively. BI/O4.1 cells are able to present various protein antigens to antigen-specific CD4 + T cells. However, a 10-fold higher concentration of antigen is needed to activate specific T cells to lymphokine secretion by BI/O4.1 cells in comparison with spleen cells or with the more homogeneous population of bone marrow-derived macrophages (BMMph). The authors tested whether the reduced antigen presentation potential of BI/O4.1 cells was augmented by transferrin-mediated uptake of the model antigen ovalbumin (OVA) coupled to human ferric transferrin. It was shown that 240-fold less OVA was sufficient to induce proliferation of an OVA-specific T-cell clone when the conjugate and not native OVA was used. The presence of ferric TF in the cultures competitively inhibited this effect of the conjugate. A similar shift in the dose-response curve to lower doses of antigen was induced by the conjugate when B lymphoma cells were used as antigen-presenting cells. BMMph and P388D1 cells processed and presented the conjugate with similar efficiency as native OVA, although both cell types exposed transferrin receptors. These data suggest that the reduced antigen presentation potential of BI/O4.1 T clone cells is due to the inefficient uptake of OVA by pinocytosis and delivery into the processing compartment.

Animals↗
PubMed2001

Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.

Alkylation↗