Preparation of suspensions of pancreatic islet cells: a comparison of methods

A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.     

The effect of chemical facilitators on the frequency of electrofusion of tobacco mesophyll protoplast

Trypsin, pronase, protease, dispase, spermine, spermidine, and DMSO were characterized for their effect on the frequency of electrofusion of tobacco mesophyll protoplasts. Protease (Boehringer Mannheim) and Sigma pronase (1.26 mg/ml; 15 min incubation) increased the fusion frequency from 7% (control) to 20.7% (2.9X increase). Following protease and pronase were trypsin (2.8X), spermine (2.4X), dispase (2.1X), DMSO (2.0X), and spermidine (1.4X). BM Protease and polyamines caused the least amount of damage, followed by DMSO and trypsin (26% and 24% decrease in viability respectively), pronase (41%) and dispase (57%). Callus formed from all but dispase-treated protoplasts. Shoots regenerated from calli of all but trypsin-treated protoplasts.Abbreviations BA 6-Benzylaminopurine - BM Boehringer Mannheim - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - FM protoplast fusion medium - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, sodium salt - MES 2-(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid - PEG polyethylene glycol - SE standard error of the mean     

Collagenase as an agent for dissolving the zona pellucida of hamster and mouse oocytes.

Crude preparations of collagenase, which have been used commonly for tissue dissociation, contain proteases that dissolve zonae pellucidae of hamster and mouse oocytes without reducing the ability of the oolemma to fuse with spermatozoa. This gentle proteolytic removal of zona is particularly useful for the study of sperm-oocyte fusion in mice, as trypsin, chymotrypsin and pronase damage the mouse oolemma.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Protection of actin against proteolysis by complex formation with deoxyribonuclease I

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-57 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.     

黄牛表皮细胞分离与体外培养最适条件的探索

为了建立黄牛表皮细胞分离与体外培养的最适条件,比较了组织块法与单细胞悬液法、不同蛋白酶(胰蛋白酶和分离酶)的消化以及有无血清培养基对细胞生长的影响,以克隆形成率来检测细胞生长和存活情况。结果证明：采用分离酶分离表皮细胞进行无血清培养是黄牛表皮细胞体外培养的最适条件。     

Reversible inhibition of protein synthesis in HeLa

Protein synthesis in suspended HeLa S₃ cells is inhibited by more than 50% immediately after addition of 100 μg pronase/ml or 500 μg trypsin/ml. Polyribosome profiles are not altered by exposure of cells to 1 or 2 mg trypsin/ml suggesting that the inhibition affects peptide chain elongation. Protein synthesis resumes after removal of proteases by sedimentation and resuspension of the cells.     

Proton pumping and oxidase activity of thermophilic cytochrome oxidase remain after its extensive proteolysis

A proton-pumping heme aa3-type cytochrome oxidase purified from the thermophilic bacterium PS3 was treated with trypsin, thermolysin, chymotrypsin, subtilisin, or pronase. The cleavage of the oxidase subunits and the effects of their cleavage on the oxidase activity and proton-pumping in reconstituted vesicles were studied. Trypsin and thermolysin cleaved some of the oxidase subunits without affecting the proton-pumping, but subtilisin and pronase cleaved all the subunits resulting in partial decrease in both activities. Chymotrypsin had an intermediate effect. Subunit II of this enzyme contains heme c which is also cleaved by proteases.     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the major protein components of the virion: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently removed HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA₂ 185–211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a major site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM β-mercaptoethanol than in the presence of 50 mM. However, the complete removal of ectodomains of HA by the high-and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein M1. The action of trans-epoxysuccinyl-L-leucylamido)(4-guanidino)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl₂ On the hydrolysis of proteins HA and M1 by bromelain was investigated.     

Proteolysis of spectrin by trypsin and pronase in the presence of phospholipid suspensions

The effect of phospholipid suspensions on the proteolysis of isolated spectrin was examined by SDS-polyacrylamide gradient gel electrophoresis. Proteolysis of spectrin in the membranes by trypsin and pronase was also studied. It was found that electrophoretic patterns of spectrin fragments were influenced by the presence of the suspension prepared from phosphatidylethanolamine:phosphatidylserine (60:40) mixture and of phosphatidylcholine. Qualitative changes in the proteolytic patterns obtained after proteolysis of spectrin by pronase in the presence of phosphatidylcholine suspension were observed. The changes in the sensitivity of spectrin towards proteases result probably from changes in the accessibility of some peptide bonds upon the interaction of this extrinsic protein with phospholipids.     

Green fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions

The green fluorescent protein (GFP) is highly resistant to proteolysis and remains uncleaved after prolonged incubation with trypsin or pronase despite several putative tryptic and chymotryptic sites in exposed loops. We have rendered GFP sensitive to proteolysis by inserting five amino acids, IEGRS, in loops at position 157, 172, or 189. Excitation and emission maxima of the three insertion mutants were similar to those of wild type, but quantum yields of mutants Omega172 and Omega189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of each mutant at a unique site defined by the insert. Pronase also yields similar digestion patterns in these variants, but further proteolysis was also observed, suggesting that the primary cleavage relaxes GFP structure and reveals previously inaccessible sites. Fluorescence of Omega189 changed little upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other variants was not affected significantly by the proteases, further confirming the remarkable stabilities of GFP variants. These constructs define a new conformation-sensitive site around residue 189 of GFP and show that GFP may be useful for design of protease-susceptible molecules for monitoring of specific proteolytic activities in vivo.     

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain

Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.     

Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme

Pro-transglutaminase from Streptomyces mobaraensis was expressed in Escherichia coli as a fusion protein carrying a C-terminal histidine tag (pro-MTG-His₆). The recombinant organism was cultivated in 15 L bioreactor scale and pro-MTG-His₆ was purified by immobilized metal affinity chromatography. Activation of the inactive pro-enzyme using trypsin resulted in an unexpected degradation of the transglutaminase and a concomitant loss of activity. Therefore, a set of commercially available proteases was investigated for their activation potential without destroying the target enzyme. Besides trypsin, chymotrypsin and proteinase K were found to activate but hydrolyze the (pro-MTG-His₆). Cathepsin B, dispase I, and thrombin were shown to specifically hydrolyze pro-MTG-His₆ without deactivation. TAMEP, the endogeneous protease from S. mobaraensis was purified for comparison and also found to activate the recombinant histidine-tagged transglutaminase without degradation. The TAMEP activated MTG-His₆ was purified and characterized. The specific activity (23 U/mg) of the recombinant histidine-tagged transglutaminase, the temperature optimum (50 °C), and the temperature stability (t_1/2 at 60 °C = 1.7 min) were comparable to the wild-type enzyme. A C-terminal peptide tag did neither affect the activity nor the stability but facilitated the purification. The purification of the histidine-tagged protein is possible before or after activation.     

Flu virion as a substrate for proteolytic enzymes

The proteolysis of flu virions of the strain A/Puerto Rico/8/34 (subtype H1N1) by enzymes of various classes was studied to develop an approach to the study of the structural organization and interaction of the basic protein components of the virion environment: hemagglutinin (HA), transmembrane homotrimeric glycoprotein, and matrix protein M1 forming a layer under the lipid membrane. Among the tested proteolytic enzymes and enzymic preparations (thermolysin, trypsin, chymotrypsin, subtilisin Carlsberg, pronase, papain, and bromelain), the cysteine proteases bromelain and papain and the enzymic preparation pronase efficiently deleted HA ectodomains, while chymotrypsin, trypsin, and subtilisin Carlsberg deleted only a part of them. An analysis by MALDI TOF mass spectrometry allowed us to locate the sites of HA hydrolysis by various enzymic preparations. Bromelain, papain, trypsin, and pronase split the polypeptide chain after the K177 residue located before the transmembrane domain (HA2 185-211). Subtilisin Carlsberg hydrolyzed the peptide bond at other neighboring points: after L178 (a basic site) or V176. The hydrolytic activity of bromelain measured by a highly specific chromogenic substrate of cysteine proteases Glp-Phe-Ala-pNA was almost three times higher in the presence of 5 mM beta-mercaptoethanol than in the presence of 50 mM. However, the complete removal of exodomains of HA, HA, and low-activity enzyme by the HA high- and low-activity enzyme required identical time intervals. In the absence of the reducing reagent, the removal of HA by bromelain proceeded a little more slowly and was accompanied by significant fragmentation of protein Ml1. The action of trans-epoxysuccinyl-L-leucylamido)butane (E-64), a specific inhibitor of cysteine proteases, and HgCl2 on the hydrolysis of proteins HA and M1 by bromelain was investigated.     

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papa?n, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.     

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).     

Topographic Distribution of Enzymes Synthesizing Phosphatidylcholine and Phosphatidylethanolamine in Chicken Brain Microsomes

Abstract: The localization of phosphatidylethanolamine and phosphatidylcholine biosynthetic enzymes within the transverse plane of chicken brain microsomes was investigated by using proteases (trypsin and pronase) and neuraminidase. Treatment of intact microsomes with the proteases inactivated the phosphocholine transferase completely and the ethanolamine phosphotransferase only slightly. This latter enzyme was, however, completely inactivated when deoxycholate-treated microsomes were exposed to proteases. Treatment of intact microsomes with neuraminidase had no effect on both phosphotransferases, although 65% of the sialic acid of sialoglycoproteins and 37% of that of gangliosides were removed. With deoxycholate-disrupted microsomes nearly all sialic acid from the sialoglycoproteins and about 70% of that of gangliosides were released. In parallel, the phosphoethanolamine transferase was 90% inactivated. It is suggested that phosphocholine transferase is localized on the outer face of the microsomal vesicle, whereas the phosphoethanolamine transferase could be a sialoglycoprotein, possibly situated on the inner face of the vesicle, or perhaps a transmembrane protein.     

Structural studies on the organization of proteins in mitochondrial membranes using proteolytic enzymes

Proteolytic enzymes, pronase and trypsin, digest protein in ETP and in SU-particles (devoid of the soluble ATPase) at similar rates and to the same extents for intact and lipid-depleted membranes, showing that lipids do not constitute a barrier to the action of the proteases. The rates and extents of hydrolysis are slightly depressed when membranes are reconstituted from lipid-depleted particles and phospholipids. The hydrolysis rates for the various particles are not greatly enhanced by detergent solubilization nor by other denaturing treatments, indicating that the rates measured in absence of treatments are maximal under the conditions used. The circular dichroism spectra of pronase treated ETP are noticeably altered showing modification of the original conformation. Moreover, enzymic activities of mitochondria and submitochondrial particles are progressively affected by proteases according to their localization at, or near to, a given surface of the membrane. The matrix enzyme, malate dehydrogenase, is not apparently released from mitochondria during the initial incubation period. The results are tentatively discussed in terms of organization of lipids and proteins in the mitochondrial membrane.     

Solubilization of porcine zonae pellucidae by trypsin and pronase

The porcine zona pellucida was dissolved with difficulty by trypsin in isotonic solution, whereas it was efficiently dissolved by pronase. A structural change of the zona was induced in hypotonic solution, resulting in acceleration of dissolution by these proteases. The solubilization rate of three families (PZP1-3) of zona protein by both enzymes was analyzed by HPLC. In hypotonic solution, PZP1 was solubilized first, followed by PZP2; and PZP3 was then finally released. In isotonic solution, PZP1 and PZP2 were also solubilized faster than PZP3, which was almost completely resistant to trypsin, showing that the solubilization of the zona depended on that of PZP3. Noticeably, high molecular weight products were produced as the proteolytic hydrolysis proceeded in PBS. Circular dichroic spectra and electrophoretograms of the tryptic hydrolysates showed that the zona may have a regular supramolecular structure.