Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Type II alveolar cells can be isolated and partially purified from adult rat lung by a series of steps that includes enzymatic digestion of the lung with trypsin and separation of cells on a discontinuous albumin density gradient. The yield of the isolated type II cells depends on the supplier and the housing of the rats used to prepare the cells. With specific pathogen-free rats housed in a laminar flow hood, the yield was 20.3 x 10(6) cells per rat, of which 50 per cent were type II cells. With rats from 2 other suppliers and no special housing, the yields were 8.8 and 8.3 x 10(6) cells per rat, of which 67 and 65 per cent were type II cells. The ultrastructural appearance of the isolated cells was similar to that of cells from intact lung, except for some dilatation of the endoplasmic reticulum and the perinuclear space. Most cells (92 +/- 5 per cent) excluded the vital dye, trypan blue. The cells consumed O2 at the rate of 76 +/- 12 nmole per 10(6) cells per hour and released only 5.7 +/- 2.0 per cent of their lactate dehydrogenase, a cytoplasmic enzyme, into the medium after 1 hour of incubation. The isolated type II cells contained disaturated phosphatidylcholine, a major component of purified surface-active material. The cells, however, had a low glucose utilization compared to their O2 consumption, which may indicate an abnormality in the metabolism of glucose. This population of cells could be further purified to 89 per cent type II cells by unit gravity velocity sedimentation.
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PMID:Isolation and properties of type II alveolar cells from rat lung. 26 96

A latent ATP-dependent Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. The data indicate that a fraction of the Ca uptake (measured with 45Ca) is associated with the intraterminal mitochondria; it is blocked by ruthenium red, by FCCP, and by azide + dinitrophenol + oligomycin. There is, however, a residual ATP-dependent Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondrial) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. The proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the ATP-dependent Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca2+ concentration (buffered with EGTA) is less than 0.3 micrometer; brain mitochondria take up little Ca when the ionized Ca2+ level is this low. Preliminary subfractionation studies indicate that the nonmitochondrial Ca storage system does not sediment with synaptic vesicles. We propose that this Ca storage system, which has many properties comparable to those of skeletal muscle sarcoplasmic reticulum, may be associated with intraterminal smooth endoplasmic reticulum. This Ca-sequestering organelle may help to buffer intracellular Ca.
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PMID:Calcium buffering in presynaptic nerve terminals. I. Evidence for involvement of a nonmitochondrial ATP-dependent sequestration mechanism. 35 58

Antibodies against the purified fragment of cytochrome b(5) released by trypsin treatment from rat liver microsomes were raised in rabbits and used for the demonstration of membranes rich in cytochrome b(5), in particular the endoplasmic reticulum (ER) system, by indirect immunofluorescence microscopy. The method allowed the demonstration of ER not only in frozen sections of various tissues, including liver and lactating mammary gland from different species, but also in cultured cells of a diversity of species and cell types. In the cultured cells the structures most prominently decorated with the antibodies against cytochrome b(5) were the nuclear envelope and the ER system which in many cells could be recognized as a system of smoothly bending, branching threads, extending from the perinuclear cytoplasm toward the cell periphery. Changes in the display of such elements during mitosis and cell plating and possible influences of the specific fixations used are discussed.
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PMID:Demonstration of the display of components of the endoplasmic reticulum system by indirect immunofluorescence microscopy using antibodies against cytochrome b(5) from rat liver microsomes. 36 Dec 57

The direction of discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 from bound polyribosomes of rough microsomes was investigated in order to elucidate the mechanism of separation of these membrane proteins from secretory proteins, which are also synthesized by the same class of ribosomes of rough endoplasmic reticulum. The nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 in intact rough microsomes were accessible to externally added 125I-Fab's against these proteins, and were susceptible to trypsin digestion, whereas the nascent peptides of serum albumin were not. The nascent peptides of these two microsomal proteins were released into the cytoplasm by puromycin treatment of intact rough microsomes, while the nascent peptides of serum albumin were retained in the microsomal lumen. These observations suggest that the nascent peptides of microsomal proteins, which are present on the cytoplasmic surface of the endoplasmic reticulum membrane, are exposed on the surface of microsomal vesicles, while those of secretory proteins are enclosed inside the vesicles. Therefore, the topographical separation of microsomal membrane proteins from secretory proteins is accomplished at the step of their synthesis by the bound polyribosomes of rough endoplasmic reticulum.
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PMID:Biogenesis of endoplasmic reticulum membrane in rat liver cells. II. Discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 on the cytoplasmic side of the endoplasmic reticulum membrane. 41 27

Distributions of parathyroid hormone (PTH), proparathyroid hormone (ProPTH), preproparathyroid hormone (PreProPTH), and parathyroid secretory protein (PSP) were analyzed in subcellular fractions prepared from homogenates of bovine parathyroid glands. Slices of bovine parathyroid glands were incubated with radiolabeled amino acids for 3--30 min to selectively label newly synthesized proteins. Subcellular fractions were prepared from homogenates of the gland slices by differential centrifugation. Newly synthesized labeled hormonal polypeptides in the fractions were analyzed by electrophoresis on polyacrylamide gels, and total amounts of PTH and ProPTH (previously formed and newly synthesized) were determined by immunoassay. Ninety percent of total immunoreactive, 70--80% of newly synthesized PTH, ProPTH, and PreProPTH, and 50% of PSP were found in sedimentable particulate fractions. The low speed (800 X g) pellet, which consisted predominantly of cell debris and nuclei with adherent remnants of cytoplasm, contained 30--50% of the ProPTH and PTH. The intermediate speed (10,000 X g) pellet, which contained granules, was relatively enriched in PTH. Most particulate-associated hormone could be solubilized by treatment with deoxycholate (DOC) 98% and 97% of radiolabeled and 93% and 83% of immunoreactive ProPTH and PTH, respectively, in particulates sedimenting at 10,000 and 105,000 X g were rendered DOC-soluble. Approximately 50% of the PTH and ProPTH in the particulates resisted digestion by combined trypsin and chymotrypsin, whereas PreProPTH was completely susceptible to proteolysis. Up to 50% of the radiolabeled PTH and ProPTH added exogenously to parathyroid gland slices before homogenization became associated with the particulate fractions, and 70--80% or radiolabeled PreProPTH added to the subcellular fractions readily associated with the sedimentable material. The results indicate that in homogenates of parathyroid glands, PTH, ProPTH, PreProPTH, and PSP are associated with particulate structures. Furthermore, up to 50% of the association of ProPTH, PTH, and PSP with particulate fractions seems to be nonsepcific and occurs during the disruption of the tissues. The remaining 50% or more of hormonal protein is presumably sequestered within membrane-limited structures, such as microsomal vesicles. The complete susceptibility in particulate fractions of newly synthesized PreProPTH, but not of ProPTH, to limited proteolysis indicates that the two precursors are located in different subcellular compartments and suggests that PreProPTH is converted to ProPTH before its entry into the intracisternal space of the endoplasmic reticulum. Alternatively, the PreProPTH identified in parathyroid gland slices may represent polypeptide chains synthesized in the cell sol on polyribosomes that are not attached to endoplasmic reticulum but are adsorbed nonspecifically to the particulate fraction of the cell during the process of tissue homogenization.
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PMID:Subcellular distributions of parathyroid hormone, hormonal precursors, and parathyroid secretory protein. 44 53

Injury in the periphery of the lung was studied in rats given intratracheal trypsin. Mitotic figures were noted in the alveolar epithelium, and colchicine enhanced the yield. Of 65 mitotic figures examined in detail, 46 were present in the alveolar epithelium. Epithelial mitoses were confined to type II cells, many of which contained lamellar inclusions, abundant rough endoplasmic reticulum, and luminal microvilli. Mitoses were not seen in type I cells or in less well differentiated epithelial cells, but they were noted in occasional endothelial and interstitial cells. Colchicine-arrested type II cells were distinguished by radially disposed chromosomes surrounding pairs of centrioles and randomly oriented microtubules. In addition, many of these cells showed a pronounced cortical zone, devoid of microvilli, and others showed cytotoxic degenerative changes. These observations indicate that the alveolar epithelium is renewed exclusively by division of differentiated type II cells.
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PMID:Mitoses in peripheral pulmonary epithelium: the effect of colchicine. 47 12

Myxoviruses (ortho- and paramyxoviruses) possess on their surface two virus-specific glycoproteins, the functions of which are largely understood; These glycoproteins are synthesized on the rought endoplasmic reticulum, and during their transport to the plasma membrane on smooth intracellular membranes, they undergo modification through proteolytic cleavage. In this way, the orthomyxovirus hemagllutinin is converted from a high-molecular weight form (HA) into two smaller cleavage products (HA1 and HA2). With the paramyxoviruses, the glycoprotein F, which is responsible for fusion and hemolysis, is derived from proteolytic cleavage of a precursor, F0. Furthermore, with a few strains of avirulent NDV, a precursor of the hemagglutinin-neuraminidase complex, (HN0), has been identified which again, as a result of proteolysis, undergoes cleavage to HN. Whether cleavage takes place is as much dependent on the structure of the glycoprotein as on the host cell type. Proteolytic cleavage is indeed not necessary for virus particle production but is required for infectivity. Virus particles which possess the uncleaved glycoproteins may be activated by in vitro treatment with trypsin. As evidenced by experiments with orthomyxovirus recombinants, the glycoproteins alone do not determine the pathogenicity of the viruses. With paramyxoviruses, the pathogenic and apathogenic strains show clear differences in their host range spectrum which is directly related to the sensitivity of their glycoproteins twoard proteases. These observations provide an initial sketch for the molecular basis of infectivity and pathogenicity with myxoviruses.
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PMID:[Correlation between structure and pathogenicity of myxoviruses (author's transl)]. 57 26

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to examine the polypeptide patterns of rat liver rough and smooth endoplasmic reticulum (ER) membrane fractions stripped of ribosomes. Approximately 67 polypeptides were resolved from the rough ER membrane fraction. The polypeptide pattern of the smooth ER membrane fraction was similar to that of the rough ER membrane fraction, but exhibited substantially lower amounts of some seven polypeptides. Three of these polypeptides, of apparent molecular weights 63,000, 65,000, and 87,000, were of particular interest, as they could not be ascribed to contamination of stripped rough ER membrane fractions by residual ribosomal polypeptides. Conditions of treatment with low concentrations of trypsin were established that markedly diminished the capacity of the stripped rough ER membrane fraction to bind ribosomes in vitro and that also effected a partial detachment of ribosomes from nonstripped rough ER membranes; the results of electrophoretic analyses of rough ER membrane fractions treated in these manners are described. Comparison of the polypeptide patterns of guinea pig, mouse, and rabbit liver ER membrane fractions with rat liver ER membrane fractions revealed considerable variations in the distribution of the polypeptides of 63,000, 65,000, and 87,000 molecular weight among the ER membrane fractions of these species. The combined results of these studies indicate that the polypeptide of 87,000 molecular weight, although particularly sensitive to attack by trypsin, is not involved in the binding of ribosomes to the rough ER membrane fraction. Studies by others (cf. Kreibich, G., Grebenau, R., Mok, W., Pereyra, B., Rodriguez-Boulan, E., and Sabatini, D. D. (1977) Fed. Proc. 36, 656) have implicated the polypeptides of 63,000 and 65,000 molecular weight in this process. The patterns of phosphorylated polypeptides of rough and smooth ER membrane fractions of rat and mouse liver were also examined, using labeling in vivo with sodium [32p]phosphate or in vitro with [gamma-32P]ATP. Approximately 25 phosphorylated components were resolved by electrophoresis in the ER membrane fractions of both species. Evidence is presented that suggests that the great majority of these components are phosphopolypeptides. Differences were noted in the patterns of phosphorylation produced by in vivo and in vitro labeling; minor differences were also observed between the patterns of phosphorylation of the rough and smooth ER membrane fractions in either situation. The overall results afford an indirect approach toward evaluating the possible involvement of specific rough ER membrane polypeptides in ribosome-binding and reveal that liver ER membranes contain a substantially greater number of phosphorylated polypeptides thatn previously reported.
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PMID:Electrophoretic studies on liver endoplasmic reticulum membrane polypeptides and on their phosphorylation in vivo and in vitro. 63 54

Specific binding of [3H]prostaglandin E2 ([3H]PGE2) and [3H]prostaglandin F2 alpha ([3H]PGF2 alpha) to plasma membrane and smooth endoplasmic reticulum fractions prepared from rat skin was demonstrated by the Millipore filter assay system. Specific binding was greater in the smooth endoplasmic reticulum fraction. Maximum binding was attained in the presence of Ca++ (0.5 x 10(-3) M), pH 7.2, and at a temperature of 37 C. Unlabeled PGE2 at a concentration of approximately 700 x 10(-9) M inhibited binding to the smooth endoplasmic reticulum fraction by approximately 90%. Other unlabeled prostaglandins, PGE1, PGF2 alpha, and a PGE2 analog, 7-0-13-prostynoic acid, at the same concentration inhibited binding by 40%, 25%, and 30%, respectively. A variety of unlabeled fatty acids (palmitic, oleic, linoleic, eicosatrienoic, and eicosatetraenoic acids), at the higher concentration of approximately 1700 x 10(-9) M, inhibited binding less than 30%, suggesting the presence of specific receptors for PGE2 in the smooth endoplasmic reticulum. Similar results were obtained for the competitive binding studies with [3H]PGF2 alpha. Proteolytic digestion of the membrane fraction by trypsin and pronase, or boiling for 15 min caused marked inhibition of binding, suggesting that the receptors for PGE2 and PGF2 alpha have a protein component. The Scatchard plot analysis of the equilibrium-binding data of [3H]PGE2 and [3H]PGF2 alpha to normal smooth endoplasmic reticulum fractions revealed an apparent dissociation constant (Kd) of 1.1 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 1,0 x 10(-9) M with a binding site concentration of 35 x 10(-12) M for [3H]PGF2 alpha. These data indicate a greater concentration of binding sites for PGE2 than PGF2 alpha in the normal skin smooth endoplasmic reticulum. On the other hand, analysis of smooth endoplasmic reticulum from skin of essential fatty acid-deficient rats revealed a Kd of 1.2 x 10(-9) M with a binding site concentration of 75 x 10(-12) M for [3H]PGE2, and a Kd of 2.2 x 10(-9) M with a binding site concentration of 175 x 10(-12) M for [3H]PGF2 alpha. The concentration of binding sites for PGF2 alpha in the smooth endoplasmic reticulum of the skin of these rats is increased 5-fold when compared to normal values, whereas that of PGE2 was not altered. These results suggest a possible alteration of PGF2 alpha specific binding to skin endoplasmic reticulum during the pathophysiological abnormalities that accompany essential fatty acid-deficiency syndrome.
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PMID:Specific binding of prostaglandins E2 and F2alpha by membrane preparations from rat skin. 74 27

Two enzymes with tyrosinase activity have been purified from the frog Rana pipiens. Both enzymes are isolated in an inactive form which can be activated with trypsin. Amino acid analysis, NH2-terminal amino acid determination (arginine for both proteins), and immunological evidence indicate that athe two enzymes are similar if not identical. They can be distinguished by their trypsin activation kinetics. Cell fractionation studies suggest that one form is found associated with the smooth endoplasmic reticulum whereas the other protein fraction is localized mainly within the premelanosomes. Sedimentation equilibrium studies demonstrate that both protein fractions are self-associating systmes. Ionic strength, temperature, and specific anion effects alter the equilibria of the associating systems. The monomeric molecule weight for both fractions is 30,000 and at low ionic strengths the predominant molecular weight species is the tetramer. The partial specific volume of each protein is 0.70.
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PMID:Tyrosinases in Rana pipiens. Purification and physical properties. 80 96


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