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PubMed1999

Pronase destroys the lipopolysaccharide receptor CD14 on Kupffer cells.

CD14 is a lipopolysaccharide (LPS) receptor distributed largely in macrophages, monocytes, and neutrophils; however, the role of CD14 in activation of Kupffer cells by LPS remains controversial. The purpose of this study was to determine if different methods used to isolate Kupffer cells affect CD14. Kupffer cells were isolated by collagenase (0.025%) or collagenase-Pronase (0.02%) perfusion and differential centrifugation using Percoll gradients and cultured for 24 h before experiments. CD14 mRNA was detected by RT-PCR from Kupffer cell total RNA as well as from peritoneal macrophages. Western blotting showed that Kupffer cells prepared with collagenase possess CD14; however, it was absent in cells obtained by collagenase-Pronase perfusion. Intracellular calcium in Kupffer cells prepared with collagenase was increased transiently to levels around 300 nM by addition of LPS with 5% rat serum, which contains LPS binding protein. This increase in intracellular calcium was totally serum dependent. Moreover, LPS-induced increases in intracellular calcium in Kupffer cells were blunted significantly (40% of controls) when cells were treated with phosphatidylinositol-specific phospholipase C, which cleaves CD14 from the plasma membrane. However, intracellular calcium did not increase when LPS was added to cells prepared by collagenase-Pronase perfusion even in the presence of serum. These cells were viable, however, because ATP increased intracellular calcium to the same levels as cells prepared with collagenase perfusion. Tumor necrosis factor-alpha (TNF-alpha) mRNA was increased in Kupffer cells prepared with collagenase perfusion 1 h after addition of LPS, an effect potentiated over twofold by serum; however, serum did not increase TNF-alpha mRNA in cells isolated via collagenase-Pronase perfusion. Moreover, treatment with Pronase rapidly decreased CD14 on mouse macrophages (RAW 264.7 cells) and Kupffer cells. These findings indicate that Pronase cleaves CD14 from Kupffer cells, whereas collagenase perfusion does not, providing an explanation for why Kupffer cells do not exhibit a CD14-mediated pathway when prepared with procedures using Pronase. It is concluded that Kupffer cells indeed contain a functional CD14 LPS receptor when prepared gently.

Animals↗
PubMed1986

[Mechanism of action of pronase on chronic proliferative inflammation including granuloma angiogenesis in mice].

Effects of pronase on granuloma angiogenesis and granulation tissue formation were investigated using granuloma pouches induced by Freund's complete adjuvant containing 0.1% croton oil in normal and alloxan diabetic mice. Granuloma angiogenesis was measured by the carmine dye infusion method. Pronase (2 mg/kg/day) was daily injected into the pouch for 3 to 7 days, and its effects were compared with those of hydrocortisone (1 mg/kg/day) and indomethacin (2 mg/kg/day). Pronase significantly inhibited the wet weight of pouch granuloma, mainly in the later stage of inflammation in normal mice. But it failed to decrease both the content of hydroxyproline in granuloma and the amount of carmine dye infused into the newly developed capillaries, whereas hydrocortisone and indomethacin significantly decreased them. In pathohistological findings, pronase increased angiogenesis and subsequently enhanced the repair rate by a dense arrangement of mature connective tissue, although hydrocortisone and indomethacin markedly decreased angiogenesis. In alloxan diabetic mice, granulation tissue was significantly produced less than that in normal mice. Hydrocortisone potentiated the decrease in granulation tissue formation of diabetic mice, whereas pronase did not markedly affect on it. The ratios of the amount of capillaries to the wet weight of granuloma tissue in pronase-treated mice were higher than those of control mice. Pronase was concluded to enhance angiogenesis in granuloma tissue in addition to the activation of collagenolysis and proteolysis.

Animals↗
PubMed2007

Premedication with pronase or N-acetylcysteine improves visibility during gastroendoscopy: an endoscopist-blinded, prospective, randomized study.

AIM: To assess the efficacy of premedicaton with pronase or N-acetylcysteine (NAC) at 20 min before upper gastrointestinal (UGI) endoscopy and to determine whether pronase or NAC pretreatment influences the reliability of the rapid urease test. METHODS: A total of 146 patients were prospectively and randomly assigned into the study groups according to different premedications before endoscopy. One endoscopist assessed mucosal visibility (MV) with scores ranged from 1 to 4 at four sites in the stomach. The sum of the MV scores from these four locations was defined as the total mucosal visibility (TMV) score. Identification of H pylori was performed using CLO test, histology, and serology. RESULTS: The Group with pronase premedication had a significantly lower TMV score than did the groups with gascon and gascon water (P < 0.001 and P < 0.01, respectively). The group with NAC had a significantly lower TMV score than the group with gascon (P < 0.01) and a trend of a lower MV score than the group with gascon water (P = 0.06). The TMV score did not significantly differ between the group with pronase and the group with NAC (P = 0.39 and P = 0.14, respectively). The sensitivity and specificity of the CLO test were 92.5% and 93.9%, respectively, in groups premedicated with pronase and NAC together. CONCLUSION: Premedication with pronase or NAC at 20 min before UGI endoscopy improves the mucosal visibility of the stomach. Neither pronase nor NAC produces any obvious interference with the CLO test for the identification of H pylori infection.

Acetylcysteine↗
PubMed1983

Role of pronase-resistant peptide segments of the antitumor protein antibiotics, auromomycin and macromomycin, in stabilizing cytocidal activity of the chromophore moieties to carcinoma cells.

The free chromophores isolated from the antitumor protein antibiotics, auromomycin (AUR) and macromomycin (MCR), were rapidly inactivated by incubation in serum-containing medium at 37 degrees C in the dark with respect to cytocidal activity to human lung carcinoma A549 cells. Under the same conditions, the intact antibiotics, their pronase-hydrolysates and reconstituents from the chromophores and apo-proteins were stable. Intact and reconstituted AUR and MCR were more resistant to pronase digestion than the apo-proteins. The analyses of the pronase-hydrolysates of AUR and MCR by SDS-polyacrylamide gel electrophoresis and ultrafiltration showed that the antibiotics (13 kilodaltons (kDa] were degraded to produce peptide fragments (1-3 kDa) in which most cytotoxicity of the pronase-hydrolysates resided. The pronase-hydrolysates exhibited a differential cytocidal activity to normal diploid fibroblasts (WI38), their SV40-transformants (VA13) and carcinoma cells (A549) of human lung origin as was observed for the intact antibiotics. These results indicate that specific interaction between the chromophores and the pronase-resistant peptide segments (1-3 kDa) of the protein moiety stabilizes the cytocidal activity of the chromophores and also protects the peptide segments from pronase digestion.

Anti-Bacterial Agents↗
PubMed2002

Removal of mucus for ultrastructural observation of the surface of human gastric epithelium using pronase.

BACKGROUND: Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori-induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase. MATERIALS AND METHODS: To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM. RESULTS: We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours. CONCLUSIONS: Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori-induced gastric disorders by SEM.

Biopsy↗
PubMed2002

A defoaming agent should be used with pronase premedication to improve visibility in upper gastrointestinal endoscopy.

BACKGROUND AND STUDY AIMS: The study tested whether pronase can improve endoscopic visibility and alter the accuracy of the CLO test for H. pylori detection. PATIENTS AND METHODS: A total of 160 patients were randomly assigned to receive one of five premedications for endoscopy: group A: dimethylpolysiloxane (DMPS) alone; group B: DMPS plus water (up to 100 ml); group C: pronase only, with 100 ml water; group D: pronase and sodium bicarbonate plus water up to 100 ml; group E: pronase, sodium bicarbonate, and DMPS, plus water up to 100 ml. Endoscopists, who were unaware of the premedication method administered, assessed visibility scores (range 1 - 4) for the antrum, lower gastric body, upper gastric body, and fundus. The higher the score, the less clear the visibility. The sum of scores from the four locations was defined as the total visibility score. A CLO test was also done during the endoscopy. One week after their endoscopy, patients in groups C, D, and E were scheduled for a (13)C-urea breath test (UBT). RESULTS: Group E patients had a significantly lower total visibility score than those in the other four groups ( P < 0.05). Groups C and D had higher total visibility scores than the other three groups ( P < 0.05). The scores did not significantly differ between groups A and B. Based on the UBT results, the sensitivity and specificity of the CLO test were 92.6 % and 96.2 %, respectively. CONCLUSIONS: Premedication as in group E provided the clearest endoscopic visibility. Without the application of DMPS, pronase alone cannot improve endoscopic visibility. Pronase does not influence H. pylori identification using the CLO test.

Adult↗
PubMed1985

Proteoglycans from lungs of rabbits treated with pronase and cadmium chloride.

The composition of proteoglycans was studied in lungs from rabbits treated intratracheally with pronase, CdCl2, or saline. Proteoglycans were extracted from the lungs by 0.5 M and 4.0 M guanidine hydrochloride (GdnHCl) in sequence. Proteoglycans were isolated from the extracts by CsCl isopycnic centrifugation and fractionated by gel filtration on a Sepharose CL-4B column. The lungs from pronase-treated rabbits had approximately 33% greater total uronate than did lungs from saline-treated control animals, whereas CdCl2-treated rabbit lungs had 28% less uronate than did control lungs. Pronase-treated animal lungs had a greater proportion of hyaluronic acid and dermatan sulfate than did the control animal lungs. Relatively greater amounts of uronate were extracted by 0.5 M GdnHCl from the enzyme-treated rabbit lungs than from the CdCl2- or saline-treated animal lungs. The concentrations of hyaluronic acid and heparan sulfate in 0.5 M GdnHCl extract and dermatan sulfate in 4.0 M GdnHCl extract were greater in pronase-treated animal lungs than in control lungs. Gel filtration of proteoglycans sedimented at q greater than 1.45 revealed that pronase-treated animal lungs had proteoglycans smaller in molecular size than those in control lungs. The observations indicate that intratracheal treatment with pronase and CdCl2 alters the composition of proteoglycans in the lung, and such alterations are likely important in the pathogenesis of emphysema.

Animals↗
PubMed1990

Pronase does not reduce the protein content of Hirt supernatant of normal mouse brain.

Normal mouse brain has been used as a model in experiments to explain the reduction of infectivity obtained following incubation with Pronase of brain infected with the scrapie infective agent. Incubation of Hirt supernatants of normal mouse brain with Pronase had no effect on the protein content when compared to controls similarly incubated without Pronase. This points to a resistance of the proteins in the brain extract to protease or to the presence of anti-protease activity. Much of the 'RNA' and 'DNA' detected in the Hirt supernatant by the colorimetric procedures for these nucleic acids was alcohol soluble. The nature of the alcohol-soluble 'nucleic acid' in mouse brain and the resistance of the proteins present in Hirt supernatants to Pronase require further investigation. In experiments where Pronase reduced the infectivity of brain preparations containing the scrapie agent, the effect is probably not due to protease activity; another activity of Pronase must be involved.

Animals↗
PubMed1985

Pronase treatment of lymphocytes reduces the time required for onset of S phase in response to anti-immunoglobulin.

Murine B lymphocytes in the presence of antibody specific for surface membrane immunoglobulin begin to synthesize DNA at about the 36th hr of culture, although the onset of synthesis in response to other B cell-reactive mitogens occurs at approximately 18 hr. In contrast, the onset of DNA synthesis by Pronase-treated cells in response to anti-immunoglobulin required only 18 hr. This earlier onset of S phase was not observed when Pronase treatment was performed in the presence of ovalbumin or the protease inhibitors phenylmethylsulfonyl fluoride or aprotinin. It is unlikely that simple carryover of Pronase from the treatment procedure to the cell culture process was involved, because Pronase treatment for 1 hr at 3 degrees C rather than at 37 degrees C did not result in early onset of DNA synthesis. Cells treated with Pronase and then with mitomycin C to irreversibly inhibit their capacity to synthesize DNA were incapable of inducing early onset of S phase on co-culture with untreated cells, suggesting that Pronase may act directly on B cells rather than indirectly via other cells in the splenocyte population.

Animals↗
PubMed2005

Alzheimer paired helical filaments (PHFs) studied by high-resolution TEM: what can vertical Pt-C replication tell us about the organization of the pronase-digested PHF core?

Untreated paired helical filaments (PHFs) and pronase-digested PHF-core filaments were stereoscopically imaged with a freeze-drying vertical platinum-carbon replication preparation method for TEM. The untreated PHF have an average wide region (W) = 22.8 +/- 2.4 nm, a narrow region (T) = 10.6 +/- 1.7 nm, and a helical turn period (L) = 78.6 +/- 13.4. The surfaces of the untreated PHF's fuzzy coat appears disorganized. The widths of the pronase-treated PHF-core filaments were significantly reduced (W(d) = 14.8 +/- 1.2 nm, T(d) = 5.7 +/- 1.0 nm, and L(d) = 75.4 +/- 17 nm). The surfaces of the untreated PHF contained approximately 1.1 nm strands, the same size as tau monomer ( approximately 1.0 nm). The pronase-digested PHF cores mostly contained approximately 1.6 +/- 0.3 nm strands although strand diameters ranged from 0.6-2.5 nm. The strands sometimes appear to be wrapped around the filament axis; less often, they appear to be roughly parallel to the PHF axis, and otherwise appear to be randomly oriented. Images of pronase-digested PHF core images are discussed in relation to the core's biochemical composition, its proposed beta structure, and structural subunit models. Images of the untreated and the pronase-digested PHF support a helical ribbon morphology.

Alzheimer Disease↗
PubMed1984

Nonresponsiveness of immature B lymphocytes to anti-immunoglobulin is reversed by pronase.

Splenic B cells are induced to proliferate upon culture with antibody having specificity for surface membrane immunoglobulins. Cells treated with pronase, washed and then cultured with antibody, exhibited a greater than 5-fold enhancement of DNA synthesis whereas pronase treatment, per se, was not mitogenic. The pronase effect exhibited specificity in that the induction of proliferation with either lipopolysaccharide or dextran sulfate was not enhanced by prior enzyme treatment. Cells from mice at two weeks of age which essentially do not show a proliferative response to antibody become responsive subsequent to pronase treatment. These results are interpreted to suggest a possible growth regulatory role for the pronase sensitive surface membrane component.

Aging↗
PubMed1981

Fertilization of pronase-treated mouse ova in vitro.

In order to obtain consistently a large number of zona-free mouse ova for studies of sperm-egg interactions, a study was made of the relative effectiveness of removing the zona pellucida from ova by mechanical or enzymatic treatments. Ova exposed to pronase before mechanical removal of the zona pellucida in medium devoid of pronase had similar fertilization rates in vitro compared with ova mechanically denuded in the absence of pronase. Ova with pronase-weakened zonae were easier to denude and survived the mechanical manipulations better than the ova denuded by vigorous aspiration in narrow-bore pipettes. However, exposure to pronase did significantly lower the incidence of polyspermy in the naked ova, indicating that some of the enzyme may have diffused across the perivitelline space and damaged sperm-binding sites on the vitelline plasma membrane. The enzyme treatment also reduced the fertilization rate of zona-intact ova.

Animals↗
PubMed1974

Chemical characterization and pronase susceptibility of the Na:K pump-associated phosphoprotein of human red blood cells.

The phosphoproteins formed by incubation of red cell ghosts with [gamma-(32)P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The (32)P-labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of (32)P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the (32)P-phosphoprotein present in the gels. The molecular weight of the (32)P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na:K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts.

Adenosine Triphosphatases↗
PubMed1983

Effect of pronase treatment, microdissection, and zona pellucida removal on the development of porcine embryos and blastomeres in vitro.

The in vitro development of porcine blastomeres and the effects of pronase treatment, microdissection, and zona pellucida removal used in the isolation procedure were investigated. Seven hundred and forty-nine two to eight-cell embryos were collected from 11 sows and 74 gilts. Zona-free porcine blastomeres (ISOL BL) were obtained by treating embryos with 2.5 or 5.0% pronase for 3.0 min and microdissecting with finely drawn siliconized glass pipettes. The effect of the pronase treatment on subsequent in vitro development was evaluated by treating two to eight-cell embryos with 5.0% pronase for 3.0 min (PTD EMB). The effect of pronase treatment and microdissection on in vitro development was evaluated by microdissecting PTD EMB, leaving one blastomere bounded by the zona pellucida (BL ZP). Untreated two to eight-cell embryos were cultured as controls (CONTROLS). Embryos and blastomeres were cultured individually in microdrops of Whitten's medium with 15 mg/ml bovine serum albumin (WM + BSA) under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37 degrees C. Observations were conducted at 24-h intervals and at the cessation of division embryos were fixed, stained, nuclei enumerated, and cleavage indices assigned. Blastocysts and vesiculated embryos which developed were measured using an ocular micrometer. The incidence of blastocyst formation was greater (P less than 0.05) for ISOL BL from four-cell than from two or eight-cell embryos. The presence of the zona pellucida did not significantly affect the incidence of blastocyst formation by single blastomeres. Although ISOL BL did not develop as well as CONTROLS or PTD EMB (P less than 0.05), development of BL ZP was not significantly different from the respective PTD EMB. Blastocysts developing from blastomeres had fewer cells and were smaller than CONTROLS or PTD EMB (P less than 0.05). Although development of ISOL BL may have been impaired by the isolation procedures employed, BL ZP are capable of in vitro development comparable to their respective PTD EMB.

Animals↗
PubMed1985

Na+-Ca2+ exchange in rat brain microsomal membranes pretreated with pronase and/or SDS.

The effects of pronase and/or SDS pretreatment on Na+-Ca2+ exchange were studied in rat brain microsomal membranes. Pronase in concentrations that liberated 11% of the membrane proteins stimulated the Na+-Ca2+ exchange. When about 24% of the proteins were split off, the results did not differ from those in control experiments. When 40% or more of the proteins were solubilized, Na+-Ca2+ exchange was abolished. Pronase pretreatment did not change the Km value for Ca2+, it increased Vmax only. The effect of pronase was partially blocked by Trasylol. Neuraminidase had no effect on Na+-Ca2+ exchange. SDS pretreatment of the membranes inhibited Na+-Ca2+ exchange: when 25% of membrane proteins were solubilized with SDS, the Na+-Ca2+ exchange was abolished while the same amount of proteins split off with pronase did not change the rate of Na+-Ca2+ exchange as related to membrane proteins. Ischaemia lasting for 2-4 h or complete hypoxia which should stimulate endogenous proteinases due to the rise of free intracellular calcium did not influence the Na+-Ca2+ exchange. A decrease in Na+-Ca2+ exchange rate was observed when proteins with molecular weight between 45,000 and 20,000 were split off from the membranes. It is assumed that the Na+-Ca2+ antiporter is a polypeptide from the group of proteins within the above molecular weights.

Animals↗
PubMed1987

Deafferentation-induced alterations in the rat dorsal horn: II. Effects of selective poisoning by pronase of the central processes of a peripheral nerve.

Studies of deafferentation and regeneration, as well as studies requiring several tracing techniques, would benefit from availability of a substance that would selectively lesion the central components of a single peripheral nerve. Pronase, a combination of proteolytic enzymes, was tested for this purpose. Three weeks following microinjection of Pronase (5-25 mg) into the rat sciatic nerve, many ganglia cells in the L3-L5 ganglia were degenerated. Degeneration of primary afferents also was evident in the dorsal horn, as detected by silver Fink-Heimer methods. Patterns of terminal fields coincided with those mapped in normal rats for the sciatic nerve by using HRP transport. Ultrastructural changes were similar to those seen at 3 weeks following sciatic nerve section or rhizotomy, as described in our companion paper. However, degenerative changes following Pronase injection of the sciatic nerve were quantitatively greater than those following sciatic nerve section alone. Degenerating terminals were either electron lucent and swollen, electron dense, or filamentous with loss of vesicles. Postsynaptic dendrites, and occasionally somata, also showed signs of degeneration. Some became electron dense, others accumulated osmiophilic floccular material, but most became electron lucent and developed large membrane-bound cavities. Glial processes expanded around degenerating elements, wrapping around both terminals and dendrites. Glial sheets covered denervated dendritic and somatic spines, separating them from their terminals. Labyrinth formations of glial sheaths around debris were also found. Pronase appears to mimic the effects of mechanical destruction of primary afferents, but when compared to rhizotomy, is selective for the afferents of a single nerve, and, when compared to nerve section, produces a greater effect. Further, the substance is relatively safe for investigators compared to other toxins such as ricin.

Afferent Pathways↗
PubMed1993

High detection rates of cryptococcal antigen in pulmonary cryptococcosis by Eiken latex agglutination test with pronase pretreatment.

Two different kits for the detection of serum cryptococcal antigen in patients with pulmonary cryptococcosis were evaluated. The Eiken test (the Eiken Co., Tokyo), which uses pronase for pretreatment of serum, was compared with the Crypto-LA test (International Biological Laboratories, Cranbury, NJ), which did not use pronase prior to testing. Cryptococcal antigen was detected in 21 of 23 patients (91%) with the Eiken test and in only 10 of 23 patients (43%) with the Crypto-LA test (p < 0.01 by McNemar test). However, the sensitivity of two tests was identical without use of pronase, as both tests could detect as little as 10(4) cells/ml of Cryptococcus neoformans and 10 ng/ml of capsular polysaccharide of C. neoformans. In those serum specimens for which both tests were positive, titers were much higher for the Eiken test, but there was a statistically significant correlation between the two tests (coefficient correlation 0.79, p < 0.01). Cryptococcal antigen titer levels measured by the Eiken test correlated well with clinical courses. There was one false-positive reaction among 82 sera of non-cryptococcal patients. Pronase enhanced the sensitivity of the Eiken test, which appeared to be useful in patients with pulmonary cryptococcal disease, and its use may prevent unneeded lung biopsies.

Adult↗
PubMed1975

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics.

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.

Animals↗