Crude Bacterial Collagenase Research Articles

Biochemical characterization of the fibres of collagen and elastin in the aortic wall of SHRSP.

1. Vessel wall fragments consisting of collagen, elastin and other insoluble proteins were prepared from the aortas of 6 month old WKY and SHRSP and dead, elderly humans. 2. Prolonged incubations of these fragments with pepsin below or at 30 degrees C resulted in different amounts of insoluble materials containing similar or larger proportions of collagen and other insoluble proteins than the respective vessel fragments. The amounts of the pepsin-insoluble materials obtained from SHRSP were larger than those from WKY but were much smaller than those from elderly humans. 3. The elastins isolated from the vessel fragments were solubilized by pepsin much more effectively than the respective vessel fragments. 4. The pepsin-insoluble materials from WKY were composed of thin mesh-shaped materials, while these materials from both rats did not contain a significant number of distinctive fibrils of collagen, the materials from elderly humans did contain numerous distinctive fibrils of collagen. 5. Large fractions of both the collagen and other proteins in the pepsin-insoluble materials were solubilized by incubation with a crude bacterial collagenase below 30 degrees C or by incubation with pepsin above 40 degrees C where the triple-helical regions of the collagens were unfolded. 6. These results appear to indicate that the aortic wall of SHRSP contains larger amounts of some insoluble components that immobilize the collagen fibrils than that of WKY, but the aortic walls of elderly humans contain much larger amounts of these components than that of SHRSP.

Enzymatic harvesting of adult human saphenous vein endothelial cells: Use of a chemically defined combination of two purified enzymes to attain viable cell yields equal to those attained by crude bacterial collagenase preparations

Seeding vascular prostheses with enzymatically harvested endothelial cells can create endothelial linings that improve small-caliber prosthetic patency. But crude bacterial collagenases used for endothelial harvest contain cytotoxic nonspecific proteases and clostridial cell wall debris which might limit their clinical usefulness. We therefore compared the endothelial cell harvest efficiency of crude bacterial collagenase with that of purified bacterial collagenase alone, purified trypsin alone, and combinations of purified bacterial collagenase and trypsin using concentrations of pure collagenase equal in collagenolytic activity to the crude bacterial collagenase material. The efficiency of harvest from human saphenous vein segments was measured by a microtiter well-growth curve assay of the number of living endothelial cells capable of attachment to fibronectin and subsequent growth obtained per unit area of saphenous vein lumen. Whereas pure collagenase and purified trypsin alone both harvested less than 5% of the baseline endothelial cell density on the veins, a combination of purified collagenase and 0.01% w/v purified trypsin was found to harvest 22% +/- 10% (SD) (n = 8 veins) of the approximately 1.3 x 10(5) endothelial cells/cm2 available on normal saphenous veins. This figure was not statistically different from the harvest efficiency of 19% +/- 10% (N = 4 veins) (p greater than 0.05) obtained by use of 0.1% w/v crude collagenase alone. This result suggests that endothelial harvesting can be done with a defined mixture of pure enzymes which would be clinically preferable to presently used crude extracts of clostridial cultures as a standardized preparation for graft seeding.

Enzymatic isolation of chondrocytes from immature rabbit articular cartilage and maintenance of phenotypic expression in culture

The studies included here identify factors affecting cartilage digestion by crude bacterial collagenase (cCGN) and describe a cartilage digestion medium that maximizes both tissue digestion rate and viable cell yield. The basal digestion medium contained 100 mM NaCl, 3 mM K2HPO4, 1 mM CaCl2, 1 mM MgSO4, 10 mM NaHCO3, 60 mM sorbitol, 5 mg/ml of dextrose, 1 mg/ml of albumin, and 2 mg/ml of cCGN in 25 mM HEPES at pH 7.2. Approximately 45% of articular cartilage tissue was digested in this basal medium in 6 h at 37 degrees C, yielding 6.8 x 10(6) viable cells per g tissue digested. The addition of 30 microM tosyllysylchloromethane (TLCM) increased the fraction of tissue digested in 6 h to 68% (p less than 0.05) and doubled viable cell yields to 13.6 x 10(6) per g tissue digested (p less than 0.05). Withholding Mg, decreasing NaCl to 70 mM, and adding 30 mM KCl increased fractional tissue digestion to 81% (p less than 0.01) and doubled viable cell yield yet again (to 29.9 x 10(6) viable cells per g tissue digested). Supplementation with TLCM increased the rate of cartilage digestion and the yield of viable cells regardless of cCGN source or lot. Additional trypsin (0.25%) inhibited tissue digestion and decreased cell yield; this effect was reversible with the addition of TLCM. The cartilage digestion medium developed in these studies (low Mg with added K and TLCM) was very effective in digesting articular, scapular, rib, and growth plate cartilage, as well as in yielding a large number of viable chondrocytes. These cells grew well in culture and maintained their chondrocytic characteristics, secreting predominantly type II collagen and large macromolecular forms of chondroitin sulfate-rich proteoglycans.

68] Insulin-sensitive CAMP phosphodiesterase in rat adipose tissue

Publisher Summary This chapter discusses the insulin-sensitive camp phosphodiesterase in rat adipose tissue. The activity of cAMP phosphodiesterase is determined from the rate of conversion of [ 3 H]cAMP into [ 3 H]AMP. [Phosphodiesterase is stimulated maximally when the insulin concentration is 1–3 nM. The stimulation observed in the cell homogenate is approximately 2-fold, and that detected in fraction P-2 is usually 2.5- to 3.0-fold. Both basal and plus-insulin activities are affected by contaminants in crude bacterial collagenase and crude bovine serum albumin. Crude bacterial collagenase contains an acidic proteinase that rapidly decomposes insulin. Therefore, cells prepared by the collagenase method must be washed carefully. The insulin-dependent stimulation of phosphodiesterase is energy dependent, but the addition of an energy source is usually unnecessary. If adipocytes are homogenized in the presence of 1 mM EDTA or dithiothreitol, little insulin effect is detectable. If cells are homogenized at pH 7.4, the basal enzyme activity is slightly elevated.

Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone.

Crude bacterial collagenase is essential for the enzymatic isolation of cells from the membranous bone of neonatal mouse calvaria. We have employed the newly developed methodology of fast protein liquid chromatography (FPLC) to separate and quantify the isozymes of collagenase so that their role in the enzymatic isolation of cells might be determined. FPLC resolved as many as six protein peaks in less than 30 min using a single anion exchange column and separated collagenase isozymes into two classes. The Class I isozymes had a preference for the substrate Azocoll, a denatured collagen substrate, and the Class II isozymes had a preference for Hexapeptide, a synthetic substrate. Two preparations of chromatographically purified collagenase (CGN-A and CGN-B) were tested for their ability to release viable cells from bone. Both preparations of purified collagenase completely digested the calvaria in 120 min. The total cell yield obtained with CGN-A was 0.34 million cells per calvarium. The yield obtained with CGN-B was 1.01 million cells per calvarium. Each preparation of purified collagenase was analyzed using FPLC. CGN-A contained only Class I collagenase isozymes, whereas CGN-B contained a mixture of both Class I and Class II isozymes. The collagenase isozymes of CGN-B were separated by FPLC and then combined in a 4:1 ratio of Class II: Class I isozymes. Utilization of FPLC-separated collagenase isozymes for the cell isolation increased the total cell yield to 1.50 million cells/calvarium. We have concluded that there are many combinations of collagenase isozymes that will completely digest the extracellular matrix of bone. However, only a combination which favors the Class II isozymes will result in a low rate of cell destruction and high cell yields.

The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver. Identification of two cell-liberating mechanisms.

Crude bacterial collagenase was chromatographed on DEAE-cellulose to yield three peaks with proteolytic activity: an arginine esterase (DEAE-1), gelatinase (DEAE-2) and a caseinolytic activity (DEAE-3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII-affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The four fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05-0.1% w/v), which produced 70-80% liver digestion with approximately 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE-1 and DEAE-2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE-3, was partially successful (30-40% digestion, 25-30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE-3 used. However, by mixing certain fractions before perfusion two gelatinase-dependent, cell-releasing mechanisms were identified: (a) DEAE-3 with ZnII peak 1 and (b) DEAE-1 mixed with either DEAE-2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. At present we are attempting to determine any differences between hepatocytes produced by the two enzymic mechanisms.

Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum

The enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. Unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. The use of chromatographically purified collagenase alone is often ineffective or very slow to release cells from tissue. We have found that two enzymes are necessary and sufficient to isolate cells from neonatal mouse calvaria: purified collagenase and neutral protease. These two enzymes can be chromatographically purified on a preparative scale to yield 100 mg amounts of each enzyme. The purified enzymes can be recombined in amounts which will digest calvaria at the same rate as the crude bacterial collagenase from which they were derived. The cells that are isolated using the purified enzymes are undamaged, as indicated by the measurement of their equilibrium density on gradients of Ficoll and sodium metrizoate. Cells isolated with crude collagenase never reach an equilibrium density upon isopyknic centrifugation, whereas cells isolated with the purified enzymes reach an equilibrium density of 1.074 g/ml in 90 min.

Enzymatic isolation of cells from bone: cytotoxic enzymes of bacterial collagenase

The enzymatic isolation of cells from fetal rat calvaria is most effectively achieved with crude Clostridium histolyticum collagenase. However, this bacterial collagenase damages the cells during the digestion of the tissue. We have used cell density, as measured by isopycnic centrifugation on polysucrose gradients, as an indicator of cell damage. There are at least two enzymes in crude bacterial collagenase capable of damaging the cells in this tissue. One of these is clostripain that has been well characterized. The other cytotoxic enzyme is uncharacterized, and its effects are not evident until the clostripain activity has been inhibited by alpha-tosyl-lysyl chloromethane. The apparent activity of this second enzyme can be inhibited by withholding magnesium from the digestion medium and by increasing the potassium concentration of the digestion medium.

Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes

Hepatocytes isolated from the liver of the common goldfish Carassius auratus L. with crude bacterial collagenase maintained ATP levels for at least 2 h. Glycogenolysis was maximally activated by 1 X 10(-6) M epinephrine and 5.8 X 10(-9) M glucagon. In liver cells incubated in calcium-free buffer containing 1 mM ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, basal glycogenolysis was enhanced by the addition of 1-4 mM calcium but the elevation of cyclic AMP and glycogenolysis due to epinephrine was unaffected by calcium. The divalent cation ionophore A23187 did not alter basal or hormone-stimulated glycogenolysis. Isoproterenol was approximately as potent as epinephrine but phenylephrine was glycogenolytic only at very high concentrations. l-Propranolol competitively inhibited the increased glycogenolysis due to catecholamines but phentolamine was ineffective as a blocking agent. Isoproterenol and epinephrine stimulated glycogenolysis at lower concentrations than those required to elevate cyclic AMP accumulation. Phenylephrine was without effect on cyclic AMP. Propranolol competitively inhibited both epinephrine- and isoproterenol-stimulated cyclic AMP accumulation, but phentolamine did not block either response. Catecholamine-stimulated glycogenolysis in goldfish liver is apparently a beta-adrenergic effect. However, low concentrations of epinephrine enhance glycogenolysis without affecting total cyclic AMP.

The Preparation of a Bacterial Collagenase Containing Negligible Non-Specific Protease Activity

A three stage procedure for the purification of crude bacterial collagenase is described. The three stages were ion exchange chromatography on SP-Sephadex and DEAE-cellulose, followed by molecular sieve chromatography on Sephadex G-200. The end product was eluted from Sephadex G-200 as a single peak of absorbance at 280 nm, and a single zone of activity against gelatin. The active eluate was divided into two halves, designated fraction 1 and 2 collagenase. Their activities were greater than those of commercially available collagenases when assayed viscometrically against pepsin solubilized collagen from guinea-pig skins. The non-specific protease activities in both fractions were much less than in the commercially available purified collagenases, and fraction 1 collagenase liberated only 2.6% of a [3H]-tryptophan label from a substrate of 2 mg of labelled chick embryo proteins, after an 18 hour incubation. When polymeric collagen was incubated with fraction 1 collagenase, at a final enzyme : substrate ratio of 1:160, the collagen was digested, resulting in the loss of 99.8% hydroxyproline as dialysable material.

Synthesis and secretion of fibrinogen and albumin by isolated rat hepatocytes

In order to investigate the regulation of synthesis of some of the plasma proteins, especially fibrinogen, at the cellular level, we have chosen to work with suspensions of hepatocytes isolated by the perfusion of rat liver with crude bacterial collagenase. By adding soybean trypsin inhibitor to the collagenase and by avoiding mechanical damage, we have prepared cell suspensions that synthesize and secrete fibrinogen and albumin and that survive for longer than twenty hours. The fibrinogen secreted is clottable and shows the same pattern in acrylamide gel electrophoresis as fibrinogen purified from rat plasma. After a three hour lag, the rate of synthesis of fibrinogen, as measured by a solid-phase radioimmunoassay, continually accelerates, so that rates several fold greater than the in vivo rate have been observed after twenty hours incubation. Cycloheximide (0.1 mM) completely abolished the appearance of fibrinogen in the medium; whereas colchicine (0.3 mM) reduced the rate by 85%. Insulin and cortisol succinate enhanced fibrinogen synthesis and secretion. The albumin secretion profile differs in several respects from that of fibrinogen, reflecting differences in intracellular pool levels and probably distinct regulatory mechanisms.

Dispersion and isolation of beating cells from adult rat heart

The cells of adult rat heart ventricles were dispersed using crude bacterial collagenase. An investigation into the effect of shaking speed, stroke length, concentration of enzyme, ionic composition, and pH of the dispersing medium permitted the development of optimal conditions for obtaining beating myocytes. Approximately one-quarter of the initial protein content of ventricular chunks could be routinely recovered as single cells after such dispersion. Centrifugation through solutions of Ficoll in phosphate buffer selectively concentrated the beating myocytes and removed contaminating cells and tissue debris.

Analysis of a crosslinked peptide from calf bone collagen: Evidence that hydroxylysyl glycoside participates in the crosslink

A crosslinked, double-chained peptide has been isolated from calf bone collagen after digestion with crude bacterial collagenase. Initially, the 3H-labelled peptide was isolated from collagen that had been treated with [ 3H]-NaBH 4, but an almost identical peptide was also isolated from collagen without prior reduction. After periodate oxidation of the reduced peptide the two component chains were resolved by further chromatography. Amino acid compositions showed that the peptide probably derived from an intermolecular crosslink between a carboxyterminal sequence of the collagen molecule and a sequence near the aminoterminus that previously has been shown to be the site of a glycosylated hydroxylysine residue. The crosslinking compound in the reduced peptide, hydroxylysinohydroxynorleucine, appeared to have derived mainly by reduction with borohydride of hydroxylysinooxonorleucine, the keto-amine rearranged form of the dehydro crosslink. The remaining hydroxyl group of the crosslink, the one not derived by reduction of the keto group, appeared to be glycosylated.

Insulin-Receptor Interaction in Isolated Fat Cells: I. THE INSULIN-LIKE PROPERTIES OF p-CHLOROMERCURIBENZENE SULFONIC ACID

Abstract Suspensions of fat cells were prepared from rat epididymal adipose tissue by digestion of the tissue with crude bacterial collagenase. One portion of each suspension was exposed to p-chloromercuribenzenesulfonic acid (CMS), washed, and then incubated in Krebs-Ringer-bicarbonate buffer. A second portion, the control, was not exposed to CMS, but it was otherwise treated identically. Acceleration of glucose transport was estimated from the rate of conversion of glucose-14C to 14CO2 and 14C-labeled lipids. Inhibition of the lipolytic action of adrenocorticotropic hormone (ACTH) was evaluated by measuring (a) the cyclic 3',5'-AMP concentration of the cell suspensions and (b) the rate of release of glycerol into the incubation medium. The optimal concentration of CMS was 10-3 m and the optimal exposure time was 30 min. Glucose utilization by cells exposed to CMS was 5 to 20 times that of the control, but only 50 to 80% that of cells maximally stimulated by insulin. Most of the increased glucose utilization was due to increased glucose entry via a specific transport system since glucose utilization, when stimulated by CMS, was inhibited by 3-O-methyl glucose. Cells exposed to CMS showed a slight increase in nonspecific permeability, however, since (a) there was a slight increase in the amount of malic dehydrogenase appearing in the incubation medium, and (b) stimulation by CMS plus insulin exceeded that of insulin alone when the glucose concentration of the incubation medium was l20 mm. Incubation of CMS exposed cells in 2,3-dimercaptopropanol resulted in partial reversal of the stimulatory effect of CMS. Exposure of cells to CMS inhibited lipolysis both in the basal state and when lipolysis was stimulated by ACTH. This inhibition was overcome either by a 10-fold increase in the concentration of ACTH or by the addition of theophylline, showing that the CMS exposure had not irreversibly inhibited the lipolytic enzymes of the cell. The antilipolytic action of CMS was probably mediated through an effect on the adenyl cyclase system since the increase in cyclic 3',5'-AMP produced by ACTH was inhibited by the CMS exposure. This inhibitory effect of CMS was likewise overcome by a 10-fold increase in the ACTH concentration. We concluded that the metabolism of CMS-treated fat cells mimicked that of cells incubated with insulin in that glucose transport was stimulated and lipolysis was reversibly inhibited. Since the reaction of CMS with isolated fat cells seemed analogous to its reaction with the sulfhydryl groups of other proteins, we postulated that the insulin-like actions of CMS were mediated through a combination between CMS and certain sulfhydryl containing elements located on or near the cell surface.