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PubMed2006

Novel O-palmitoylscleroglucan-coated liposomes as drug carriers: development, characterization and interaction with leuprolide.

Polysaccharide-coated liposomes have been studied for their potential use for peptide drug delivery by the oral route because they are able to minimize the disruptive influences on peptide drugs of gastrointestinal fluids. The aim of this work was to synthesize and characterize a modified polysaccharide, O-palmitoylscleroglucan (PSCG), and to coat unilamellar liposomes for oral delivery of peptide drugs. To better evaluate the coating efficiency of PSCG, also scleroglucan (SCG)-coated liposomes were prepared. We studied the surface modification of liposomes and the SCG- and PSCG-coated liposomes were characterized in terms of size, shape, zeta potential, influence of polymer coating on bilayer fluidity, stability in serum, in simulated gastric and intestinal fluids and against sodium cholate and pancreatin. Leuprolide, a synthetic superpotent agonist of luteinizing hormone releasing hormone (LHRH) receptor, was chosen as a model peptide drug. After polymer coating the vesicle dimensions increased and the zeta potential shifted to less negative values. These results indicate that both SCG- and PSCG-coated liposomes surface and DSC results showed that PSCG was anchored on the liposomal surface. The stability of coated-liposomes in SGF, sodium cholate solution and pancreatin solution was increased. From this preliminary in vitro studies, it seems that PSCG-coated liposomes could be considered as a potential carrier for oral administration.

Animals↗

PubMed1983

In vitro and in vivo antibacterial activity of AT-2266.

AT-2266 [1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1,8-naphthyridine-3-carbo xylic acid] showed a broad spectrum of antibacterial activity against gram-positive and gram-negative microorganisms, including Pseudomonas aeruginosa. The in vitro antibacterial activity of AT-2266 was in general comparable to that of norfloxacin, but much higher than that of pipemidic or nalidixic acid. The 90% minimal inhibitory concentrations (MIC90s) of AT-2266 for P. aeruginosa resistant to gentamicin (MIC range, 25 to greater than 200 microgram/ml) and Enterobacteriaceae resistant to nalidixic acid (25 to greater than 1,600 micrograms/ml) were 3.13 and 12.5 micrograms/ml, respectively. The to nalidixic acid (25 to 1,600 micrograms/ml) were 3.13 and 12.5 micrograms/ml, respectively. The MICs of AT-2266 were only slightly affected by the addition of horse serum or sodium cholate, by the pH of the medium, and by inoculum size. AT-2266 was sodium cholate, by the pH of the medium, and by inoculum size. AT-2266 was bactericidal at concentrations near its MIC value. The 50% effective doses of AT-2266 after oral administration against systemic infections in mice were about 1/2 those of norfloxacin, about 1/10 those of pipemidic acid, and between 1/20 and 1/40 those of nalidixic acid.

Animals↗

PubMed1989

Expression in Xenopus oocytes of rat liver mRNA coding for a bile salt-dependent cholesteryl ester hydrolase.

A catalytically active bile salt-dependent cholesteryl ester hydrolase (CEH) was expressed when Xenopus oocytes were injected with rat liver mRNA. The expressed CEH activity was highly dependent on the presence of trihydroxy bile salts (cholate or one of its conjugates); maximum hydrolytic activity was observed in the presence of 10 mM sodium cholate. The expressed CEH was not activated by dihydroxy bile salts (deoxycholate and its conjugates). In the presence of 10 mM sodium cholate, the CEH activity was maximal near pH 7 but was significant between pH 6 and 8. Monospecific immune IgG raised against rat pancreatic CEH completely inhibited the CEH expressed in Xenopus oocytes. Phenylmethylsulfonyl fluoride, a serine enzyme inhibitor, was inhibitory to the expressed CEH activity, whereas p-chloromercuribenzoate (up to 5 mM), a potent thiol-blocking agent, did not significantly inhibit the expressed activity. These experiments clearly demonstrate that the liver contains an mRNA encoding a bile salt-dependent CEH activity and suggest that the uptake of pancreatic enzyme is not necessarily the source of liver CEH as has been speculated.

Animals↗

PubMed1970

Morphological changes of the small-intestinal mucosa of guinea pig and hamster following incubation in vitro and perfusion in vivo with unconjugated bile salts.

Incubation in vitro of the intestine of the hamster and guinea pig with 5 mM sodium cholate and with 2 mM sodium deoxycholate and sodium chenodeoxycholate resulted in significant morphological changes compared with control incubations. Generally, no major differences were observed between proximal and distal small intestine or between the species used. Only when guinea pig intestine was incubated with 5 mM cholate was less damage found proximally than distally. Perfusion in vivo of the intestine of the hamster and guinea pig with Krebs-Ringer phosphate results in separation of the epithelium from the lamina propria without excessive shedding of epithelial cells from villous tips. This change was also seen in specimens taken before perfusion and probably represents unavoidable trauma during handling of the intestine. In contrast to studies in vitro, regional differences are readily demonstrable with perfusion of bile salts in vivo. Dihydroxy bile salts produce more marked alterations of both proximal and distal small intestine than the trihydroxy bile salt, sodium cholate. Dihydroxy bile salts result in significantly greater alterations in proximal than in distal mucosa. When 5 mM cholate at pH 6.8 is perfused in the guinea pig, absorption occurs approximately 30 times more rapidly from distal than from proximal segments, while in proximal segments 2 mM chenodeoxycholate is absorbed approximately 15 times more rapidly than 5 mM cholate. A correlation is suggested between the morphological alteration produced in the region of the small intestine by a bile acid and the amount of bile salt passing through the cell. Furthermore, it is proposed that the ileal cells may be damaged to a lesser extent by bile acids normally found in that particular species.

Animals↗

PubMedDate not supplied

Accumulation of vinblastine into transfersomes and liposomes in response to a transmembrane ammonium sulfate gradient and their cytotoxic/cytostatic activity in vitro.

Vinblastine was encapsulated into liposomes composed from lipids dimiristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC), with cholesterol and transfersomes with sodium cholate prepared by the thin film hydration method. The percentage of vinblastine encapsulation, the stability of transfersomes and liposomes and the rate of release of encapsulated vinblastine at 37 degrees C were studied. The results showed that encapsulation of vinblastine into liposomes was higher than 98% at a drug/phospholipid molar ratio from 0.17 to 0.18, while encapsulation of vinblastine into transfersomes varied from 50% to 80% at a drug/phospholipid molar ratio from 0.05 to 0.09. The retention of drug in liposomes and in transfersomes was found to be time/dependent. The retention of drug in transfersomes compared to the liposomes was reduced due to the presence of sodium cholate which caused destabilization and reduced the main phase transition temperature Tm of the PC bilayers. The cytotoxic/cytostatic activity of the two liposome formulations and the two transfersome formulations with or without encapsulated vinblastine were examined against nine human cell lines and the parameters GI50, TGI, LC50 were estimated according to the NCI protocol. Free DPPC/sodium cholate liposomes found to exhibit strong antiproliferative activity in contrast to the other three free liposomal formulations (DPPC/cholesterol, DMPC/cholesterol, DMPC/sodium cholate). On the other hand, vinblastine encapsulated into the liposomes found to exhibit 20-fold less activity on average, in the three parameters calculate compare to the free vinblastine.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

Physicochemical properties of liposomes incorporating hydrochlorothiazide and chlorothiazide.

In an attempt to study the effect of hydrophobic drugs on liposome properties, multilamellar liposomes (MLV) consisting of phosphatidylcholine (PC) and incorporating chlorothiazide (CT) or hydrochlorothiazide (HCT), were prepared and characterized. Liposome size, surface charge, stability (in buffer, plasma and sodium cholate) and calcium-induced aggregation were studied for drug-incorporating liposomes and empty liposomes for comparison. Results show that drug incorporation affects liposome size, z-potential and stability in presence of buffer and plasma proteins. Indeed, drug-incorporating liposomes are slightly larger and have a negative surface charge, which increases with the amount of drug incorporated in the lipid membrane. The membrane integrity of drug incorporating liposomes (in absence and presence of plasma proteins) is significantly higher when compared with that of empty liposomes (for both drugs studied). On the contrary, vesicle membrane integrity in presence of sodium cholate and calcium induced vesicle aggregation, are not affected by drug incorporation. Leakage of thiazides from liposomes was demonstrated to be induced by dilution. Low amounts of thiazides (around 10-15%) are released when lipid concentration is over 0.1 mM, while further dilution increased drug leakage exponentially. Concluding, results demonstrate that the presence of HCT or CT in liposome membranes has a significant effect on main vesicle properties, which are known to influence vesicle targeting ability. Thereby, it is very interesting to continue studies in this respect, especially with more lipophilic drugs.

Antihypertensive Agents↗

PubMed2005

Remodeling of human plasma lipoproteins by detergent perturbation.

Detergent perturbation, the treatment of total human plasma lipoproteins (TLP) with sodium cholate and its subsequent removal, has been used to study lipoprotein dynamics and stability. At physiological TLP concentrations, detergent perturbation converts low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to higher-particle weight species with the concomitant release of apo A-I but not apo A-II as a lipid-poor species. Detergent perturbation of isolated HDL also releases lipid-poor apo A-I and forms larger HDL species, whereas detergent perturbation of an isolated LDL has no effect on its size. A model is presented in which detergent perturbation induces transfer of PC from metastable HDL and LDL to mixed micelles with sodium cholate. The remaining LDL and HDL are unstable because of the loss of their surface components, phospholipid and/or apo A-I, and fuse to give larger LDL and HDL particles. These effects on HDL, i.e., PC transfer, apo A-I dissociation, and particle fusion, emulate the activity of human plasma phospholipid transfer protein. Thus, detergent perturbation is a new and potentially powerful method for determining lipoprotein stability, studying the mechanisms for remodeling of plasma lipoproteins, and preparing new forms of HDL and LDL with unique interactions with lipoprotein transporters and receptors.

Apolipoprotein A-I↗

PubMed1982

Resolution and activity of adenylate cyclase components in a zwitterionic cholate derivative [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate].

Bovine brain adenylate cyclase was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium cholate, sodium deoxycholate, or these detergents plus (NH4)2SO4. The specific activity of the extract obtained with 13 mM CHAPS alone was several times those of the other detergent extracts with or without (NH4)2SO4. After solubilization with 13 mM CHAPS, gel filtration completely separated the catalytic unit (C) from the guanine nucleotide binding protein (G/F). C activity when assayed with 5 mM Mn2+ was 5 times that assayed with 10 mM Mg2+ and was unresponsive to GPP(NH)P. C activity was increased approximately 150% by GPP(NH)P in the presence of G/F extracted from human erythrocyte ghosts and approximately 100% by Ca2+ plus calmodulin in assays with Mg2+. On gel filtration and/or density gradient centrifugation, the physical properties of C from brain or AC- cells and G/F from bovine or pig erythrocytes in CHAPs were similar to those observed in other detergents. It appears that the use of CHAPS for solubilization of adenylate cyclase and separation of C and G/F may well prove advantageous in studies of the molecular interactions between the protein subunits and activators of the enzyme as well as for the initial purification of C.

Adenylyl Cyclases↗

PubMed1984

Studies in bile salt solutions. The effect of pH on the cholate and taurocholate stimulation of human milk lipase catalyzed hydrolysis of p-nitrophenylacetate.

The pseudo-first-order rate constants of hydrolysis of p-nitrophenylacetate, catalyzed by human milk lipase, have been measured in solutions of 0.01 mol dm-3 Bistris(2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3 -diol) buffer at 310.5 K, containing a range of concentrations of sodium taurocholate and sodium cholate, at pH 8.00 and of sodium cholate at pH 6.5. The effect of pH on the activity of the enzyme has been investigated and the stimulation factors of taurocholate and cholate ions and of cholic acid have been calculated to be equal to 5.3, 3.7 and 10.7, respectively. The essential residues for catalytic activity of the enzyme have ionization constants equal to 6.45-6.46 for pK1 and 8.33-8.40 for pK2. The former value is attributed to the presence of a histidine imidazolium group but the identity of the residue leading to pK2 is not proven.

Catalysis↗

PubMed1984

Solubilization and reconstitution of alpha 2-adrenergic receptors from rat and calf brain.

Sodium cholate and digitonin were used to solubilize alpha2-adrenergic receptors from rat and calf brain. Sodium cholate extracted 40-50% of the membrane protein and 25-30% of the binding capacity. Digitonin extracted only 20-30% of the membrane protein and only 10-15% of the binding capacity of the native membranes. Both detergents were removed by dialysis in the presence of phospholipids, and the solubilized protein was precipitated upon addition of poly(ethyleneglycol) and magnesium. In the solubilization/reconstitution process no purification of the alpha2-adrenergic receptor was obtained, most probably due to its inactivation by the solubilization conditions. The reconstituted protein(s) tested for binding properties, using p-[3H]aminoclonidine and/or [3H]clonidine, maintained the pharmacological profile of the native alpha2-adrenergic receptor. The potency order of various alpha2-agonists and alpha2-antagonists as well as their stereoselectivity were identical to those of the native alpha2-receptor. Specific receptor binding decreases in the presence of the guanyl nucleotides GTP or guanosine 5'-[beta, gamma-imido]-triphosphate but not ATP, thus indicating a co-solubilization of GTP regulatory components (stimulatory protein Ns or inhibitory protein Ni or both). Adenylate cyclase activity of the reconstituted preparation is stimulated threefold by sodium fluoride, suggesting the presence of both Ns-protein and the catalytic unit (C) in the reconstituted protein(s).

Adenylyl Cyclases↗

PubMed1995

Detection of hypoglycemic drugs in human urine using micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) is evaluated as a potential analytical method for the separation and detection of a series of sulfonylurea drugs used in the treatment of hyperglycemia. These drugs are often surreptitiously abused, producing extremely low blood glucose levels and symptoms indistinguishable from those associated with an insulin-secreting tumor. Separation buffer containing 50 mM sodium dodecyl sulfate (SDS) was found to be adequate for the MEKC separation of the third generation drugs (glipizide and glyburide) but not the second generation drugs (acetohexamide chlorpropamide, tolazamide, and tolbutamide). At a pH of 8.5 in the presence of 20 mM borate/20 mM phosphate and 150 mM SDS, all seven components were adequately resolved with an analysis time of 17 min. Altering the concentration of the buffering components to either 5 mM borate/5 mM phosphate or 40 mM borate alone reduced the analysis time to less than 10 min with no observable loss in resolution. A series of other micelle-forming surfactants were evaluated, and only sodium cholate provided an improvement over the SDS-based system. Optimal separation was obtained with 75 mM sodium cholate and led to complete analysis with baseline resolution of all seven components in less than 8 min. These conditions were shown to be adequate for the detection of the hypoglycemic drugs spiked into normal urine and in patients taking these drugs. The precision associated with nine consecutive injections of six samples (n = 54) was found to be acceptable with percent coefficient of variance for absolute migration times (MTabs) for all peaks averaging 0.89 with peak area and peak height being 8.49 and 8.26, respectively. The between-sample precision was found to average 0.92% for MTabs and 8.56% and 8.45%, respectively, for the relative peak area and peak height. With a detection limit for the drugs in urine (following extraction) in the 50 ng/mL range, the potential exists for an MEKC-based assay for the detection of sulfonylurea drugs in urine.

Chromatography, High Pressure Liquid↗

PubMed1972

Murein and the outer penetration barrier of Escherichia coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa.

A penetration barrier operating outside the periplasmic enzyme penicillinase was studied in an ampicillin-resistant mutant of Escherichia coli K-12. Growth in the presence of lysozyme and sublethal concentrations of ampicillin partially opened the barrier. This could be recorded as an increased penetration of penicillin G, sodium cholate, and rifampin to their respective targets. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and sodium cholate effectively impaired the barrier against penicillin and also caused leakage of penicillinase. Wild-type E. coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa also showed an increased sensitivity to cholate after treatment with penicillins. Electron micrographs showed that lysis by cholate was due to a distortion of the cytoplasmic membrane causing a leakage of protein and RNA from the cells to the medium. Physiological data indicated that the increased sensitivity to cholate induced by growth in the presence of ampicillin or lysozyme was due to effects upon the murein. This was supported by measurement of the incorporation of (3)H-diaminopimelic acid. These results indicate that the murein sacculus either is a part of the penetration barrier or is responsible for holding the structure of the outer membrane together.

Ampicillin↗

PubMed1991

Improved purification and characterization of membraneous and cytosolic inositol phospholipid-specific phospholipases C from porcine brain cortex.

A phospholipase C was solubilized and purified from membranes of porcine brain cortex. Simultaneously, a phospholipase C was purified from a cytosolic fraction of porcine brain cortex. The enrichment of phospholipase C from either fraction was about 1000-fold as determined by hydrolysis of phosphatidylinositol 4,5-bisphosphate. Phospholipases C purified from membranes or from cytosol were indistinguishable with regard to the following properties: The enzyme activities copurified with a protein of 145 kDa. The standard sedimentation coefficients (s20,w values) of the purified enzymes were 6.2 S in the absence or presence of 0.3% (w/v) sodium cholate; Stokes' radii, estimated by gel filtration on a Superose 6 HR 10/30 column in the presence of 0.3% sodium cholate, were 4.5 nm; calculated molecular masses were about 120 kDa; no significant hydrolysis of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine by preparations of purified phospholipase C was observed; adenine and guanine nucleotides affected the activity of purified enzymes in a complex manner. Thus, the enzymes purified from membraneous and from cytosolic fractions exhibited properties of the phospholipase C-beta form. The enzymes purified from either fraction required Ca2+ at a low concentration (100 nM to 10 microM) for maximal activity. The advantage of the present purification procedure is that the purified enzymes were free of phosphatidylinositol 4,5-bisphosphate 5-phosphatase, inositol 1,4,5-trisphosphate 5-phosphatase and guanine nucleotide-binding proteins after three chromatographic steps. The purified enzymes may, therefore, prove useful for studying the hormonal regulation of phospholipase C in reconstituted systems and for the preparation of [5-32P]inositol 1,4,5-trisphosphate from [5-32P]phosphatidylinositol 4,5-bisphosphate.

Animals↗

PubMed1977

[Membrane lipids and electron transfer. Effects of four detergents on NADH-ferricyanide reductase and NADH-cytochrome c reductase activities of potato tuber microsomes].

The NADH-ferricyanure reductase activity of Potato microsomes is stimulated by non ionic detergents (Triton X100 and Tween80) and is partially inhibited by ionic detergents (sodium-cholate and deoxycholate). All these four detergents progressively decreased the NADH-cytochrome c reductase in the following order: sodium deoxycholate greater than Triton X100 greater than sodium cholate greater than Tween80.

Cytochrome Reductases↗

PubMedDate not supplied

Inactivation of viruses in labile blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate detergent combinations.

Use of the organic solvent, tri(n-butyl)phosphate (TNBP), and detergents for the inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation obtained with TNBP plus Tween 80 was superior to that observed with ethyl ether plus Tween 80, a condition previously shown to inactivate greater than or equal to 10(6.9) CID50 of hepatitis B and greater than or equal to 10(4) CID50 of Hutchinson strain non-A, non-B hepatitis. The AHF recovery after TNBP/Tween treatment was greater than or equal to 90 percent. Following the reaction, TNBP could be removed from the protein by gel exclusion chromatography on Sephadex G25; however, because of its large micelle size, Tween 80 could not be removed from protein by this method. Attempts to remove Tween 80 by differential precipitation of protein were only partially successful. An alternate detergent, sodium cholate, when combined with TNBP, resulted in almost as efficient virus inactivation and an 80 percent recovery of AHF. Because sodium cholate forms small micelles, it could be removed by Sephadex G25 chromatography. Electrophoretic examination of TNBP/cholate-treated AHF concentrates revealed few, if any, changes in protein mobility, except for plasma lipoprotein(s).

Blood Proteins↗

PubMed1991

[Tyrosine protein kinase from cattle cerebral cortex: purification, characteristics, protein substrates for phosphorylation and inhibitors of activity].

Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphorylates enolase, tubulin and calpactin I as well as model substrates in the series: histone H5 greater than poly(G, T)n greater than or equal to histone H2A greater than poly(G, A, T)n, histone H4 greater than caseins, histones H1 and H2B, poly(G, A, L, T)n. The enzyme is specific for Mn2+ at the optimal concentration about 1 mM. The KmMn-ATP is 0.3 microM; Km for histone H5 and poly(G, T)n are 0.45 mg/ml and 0.06 mg/ml, respectively. The protein kinase p62 activity is inhibited by NaCl (IC50 approximately 75-100 mM) as well as by quercetin, adriamycin and lasalocid (IC50 approximately 14-34, 23 and 90 microM, respectively). It is concluded that protein kinase p62 is analogous to the c-src gene protein kinase.

Animals↗

PubMed1992

Cholate solubilization of liver microsomal membrane components which promote NADPH-supported lipid peroxidation.

NADPH-supported lipid peroxidation monitored by malondialdehyde (MDA) production in the presence of ferric pyrophosphate in liver microsomes was inactivated by heat treatment or by trypsin and the activity was not restored by the addition of purified NADPH-cytochrome P450 reductase (FPT). The activity was differentially solubilized by sodium cholate from microsomes, and the fraction solubilized between 0.4 and 1.2% sodium cholate was applied to a Sephadex G-150 column and subfractionated into three pools, A, B, and C. MDA production was reconstituted by the addition of microsomal lipids and FPT to specific fractions from the column, in the presence of ferric pyrophosphate and NADPH. Pool B, after removal of endogenous FPT, was highly active in catalyzing MDA production and the disappearance of arachidonate and docosahexaenoate, and this activity was abolished by heat treatment and trypsin digestion, but not by carbon monoxide. The rate of NADPH-supported lipid peroxidation in the reconstituted system containing fractions pooled from Sephadex G-150 columns was not related to the content of cytochrome P450. p-Bromophenylacylbromide, a phospholipase A2 inhibitor, inhibited NADPH-supported lipid peroxidation in both liver microsomes and the reconstituted system, but did not block the peroxidation of microsomal lipid promoted by iron-ascorbate or ABAP systems. Another phospholipase A2 inhibitor, mepacrine, poorly inhibited both microsomal and pool-B'-promoted lipid peroxidation, but did block both iron-ascorbate-driven and ABAP-promoted lipid peroxidation. The phospholipase A2 inhibitor chlorpromazine, which can serve as a free radical quencher, blocked lipid peroxidation in all systems. The data presented are consistent with the existence of a heat-labile protein-containing factor in liver microsomes which promotes lipid peroxidation and is not FPT, cytochrome P450, or phospholipase A2.

Animals↗

PubMed1982

Occurrence and some properties of membrane-bound neutral proteinase in the microsomal fraction of rat skeletal muscle.

Membrane-bound neutral proteinase was found in the microsomal fraction of rat skeletal muscle as assayed with heat-denatured casein as a substrate. The enzyme was solubilized from 1 M KC1-washed microsomal fraction by 1% sodium cholate containing 0.1 M NaCl, and partially purified by chromatography on a column of Sepharose CL-6B in the presence of 0.5% sodium cholate and 0.1 M NaCl. The enzyme was eluted from the Sepharose column as a single but rather broad peak at a position corresponding to a molecular weight of about 190,000. The pH optimum for hydrolysis of heat-denatured casein was about 8.0. It was inhibited to significant extents by various reagents including diisopropyl phosphorofluoridate, phenylmethanesulfonyl fluoride, N alpha-tosyl-L-phenylalanine chloromethyl ketone, N alpha-tosyl-L-lysine chloromethyl ketone, p-chloromercuriphenyl sulfonate, chymostatin, EDTA, EGTA, and o-phenanthroline. This inhibition profile suggests that the present muscle proteinase is a mixture of proteinases, such as serine proteinase and a metallo-proteinase similar to those occurring in the microsomal membranes of liver and kidney (or small intestine), respectively. Among urea-denatured proteins tested as substrates, calf thymus histone was hydrolyzed most rapidly, followed by protamine, hemoglobin, and casein.

Animals↗