Enzymatic Isolation Research Articles

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.

Plant cuticles sorb lipophilic compounds during enzymatic isolation

Abstract Plant cuticles sorb large amounts of hexadecanoic acid, octadecanoic acid and other lipophilic compounds (not identified) when incubated in cell slurries obtained by enzymatically digesting leaves or fruits. These extraneous substances cannot be removed completely and selectively after cuticle isolation, nor is it possible to prevent sorption by optimizing isolation procedures. It is, therefore, impossible to estimate amounts and composition of intracuticular soluble lipids using enzymatically isolated cuticles, as has been done in the past. Extraneous substances sorbed during isolation do not affect water permeability of the cuticles.

Isolation and characterization of single myocardial cells from the perch, Perca fluviatilis

1. 1. In applying the enzymatic cell isolation technique to the fish heart about 40% of the dispersed myocytes maintained their spindle-shaped morphology, and about half of them tolerated physiological concentration of Ca 2+ and excluded the vital dye, Evans blue. 2. 2. The length of spindle-shaped myocytes was on average 133 ± 3 μm and the maximum width was 4.2 ± 0.1 μm. The mean length of the sarcomeres was 2.1 ± 0.1 μm. The sizes of the myocytes did not vary significantly with the weights of the fish. 3. 3. Electron microscopic examinations showed typical fish myocardial cell structure; absence of transverse tubule system, a sparse network of sarcoplasmic reticulum and from a few up to eight or more myofibrils. The cells were mononuclear. 4. 4. Most of the Ca 2+-tolerant myocytes were quiescent, but the contraction in them could be induced by electric field stimulation. 5. 5. Both the spontaneous and electrically triggered contractions were of twitch type. The slowly propagating contraction waves, so-called phasic contractions common in isolated mammalian cardiac myocytes, could not be seen at all.

Rapid isolation and study of protoplasts obtained throughout the day-night cycle from leaves of Kalanchoe blossfeldiana showing different levels of crassulacean acid metabolism

A method is described for rapid enzymatic isolation of protoplasts from the Crassulacean acid metabolism (CAM) plant Kalanchoe blossfeldiana cv. Tom Thumb. Young leaves were sampled at low, middle or full CAM levels induced by increasing number of short-days (14, 31 and 49 SD). Maximum O 2 exchange in light or dark and maximum CO 2 fixation in light occur with protoplasts obtained at 1730 (end of the day) for all CAM levels. Dark CO 2 fixation, typical of CAM, is performed by protoplasts isolated in the middle of the night from plants having received at least 31 SD. Rates of dark CO 2 fixation by these protoplasts are of the same order as those of intact leaves. The capacity for O 2 exchange and CO 2 fixation increases with the level of CAM. These protoplasts retain characteristics typical of CAM, such as diurnal oscillations in phospho enolpyruvate carboxylase (EC 4.1.1.31; PEPC) capacity and malate content.

Enzymatic isolation of cells from aquatic macrophytes

Cells were isolated by enzymic digestion from a number of aquatic macrophytes and their photosynthetic activities were determined. Eichhornia crassipes (Mart.) Solms, Myriophyllum spicatum L. and M. brasiliense Cambess provided photosynthetically-active cells after digestion with commercial pectinase. Cells from emergent leaves of M. brasiliense were approximately 3 times more active than cells from submersed leaves (56.1 vs. 17.4 μmoles CO 2 mg −2 Chl h −1). Cells could be isolated from E. crassipes by grinding as well as by digestion, but the former were less active (3.1 vs. 24.2 μmoles CO 2 mg Chl h −1). Attempts to isolate cells from Hydrilla verticillata (L.f.) Royle or Potamogeton pectinatus L. were not successful.

Nucleolar changes during the first steps of experimental hepatocarcinogenesis in rats

Silver stainability of hepatocytes as an expression of nucleolar activity was studied in vivo during rat hepatocarcinogenesis. Male Wistar rats were injected with one dose of diethylnitrosamine (200 mg/kg body weight dissolved in 0.9% NaCl), followed by a selection procedure with a short exposure to 2-acetylaminofluorene in combination with a proliferative stimulus, such as the administration of CCl 4. Finally, after 1 week of a normal diet, some of the rats were treated with phenobarbital. After enzymatic isolation, the hepatocytes were silver stained; the estimation of nucleolar activity was determined by a cytomorphologic analysis of the silver-stained nuclei. It was demonstrated that during the first steps of hepatocarcinogenesis, both diethylnitrosamine, as initiator, and phenobarbital, as promotor, induce modifications of the nucleolar morphology in silver-stained hepatocytes.

Starch phosphorylase isoenzymes in mesophyll and bundle sheath cells of corn leaves

Starch phosphorylase from leaves of the C 4-plant corn ( Zea mays L.) could be separated into two peaks of activity by chromatography on DEAE-cellulose (or by (NH 4) 2SO 4 gradient solubilization). Subsequent polyacrylamide gel electrophoresis under native conditions revealed a slowly migrating phosphorylase for the peak eluting first from DEAE-cellulose and a major, fast migrating phosphorylase for the peak eluting second. The latter peak contained also two very minor bands which migrated even faster than the other two phosphorylases. When phosphorylases were extracted from mesophyll protoplasts and from bundle sheath strands (isolated by the enzymatic isolation procedure) it was the slowly migrating phosphorylase which was associated with the mesophyll cells and the major, fast migrating phosphorylase which originated from the bundle sheath cells. The findings are discussed in view of similar isoenzymes of phosphorylase reported for chloroplasts and cytosol in leaves of C 3-plants.

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.

The ability of isolated myocardial cells of the rat to contract at temperatures near freezing point

1. 1.|Single spontaneously-beating myocardial cells were prepared by enzymatic isolation from rat heart ventricles. Using a TV camera and videotape recorder, beating parameters were characterized with respect to temperature. 2. 2.|The cells obtained showed two types of contractions: (a) action-potential-coupled, fast contractions, which could be triggered electrically but sometimes also occurred spontaneously; and (b) a phasic type of contraction which occurs spontaneously as a slowly (25–200 μm s −1) proceedings wave through the cell and is not coupled to the action potential. 3. 3.|Both the spontaneous phasic and electrically-triggered fast contractions continued down to about 0°C, where the cells went into an irreversible contracture, but recovered if the temperature was raised before the contracture occurred. 4. 4.|In physically-contracting cells, the beat rate and the velocity of the contraction wave were temperature dependent showing a linear relationship on an Arrhenius plot; apparent activation energies were 31 and 54 kJ mol −1, respectively. 5. 5.|When fast contractions were triggered by electric field stimulation, the threshold of cells was reduced as temperature rose. The rheobase values showed that cell excitability decreased more steeply when the temperature fell below 20°C. This change could not be seen in the values of chronaxie, which showed a linear relationship on the Arrhenius plot within the whole temperature range used (6–35°C). 6. 6.|It is concluded that the lower temperature limit is not the same for all structural levels of the rat heart. Myocardial cells are able to contract still at the freezing point while mechanisms initiating the heart beat require higher temperatures to be functional.

Water permeability of periderm membranes isolated enzymatically from potato tubers (Solanum tuberosum L.)

The fine structure and water permeability of potato tuber periderm have been studied. Periderm membranes (PM) were isolated enzymatically using pectinase and cellulase. They were composed of, about six layers of phellem cells arranged in radial rows. The walls of phellem cells consist of cellulosic primary and tertiary walls and suberized secondary walls which are lamellated. Middle lamellae and primary walls contain lignin. Since the PM did not disintegrate during enzymatic isolation it appears that lignin also extends into the secondary suberized walls. The water permeability of PM was low, ranging from 1-3·10(-10) m s(-1). This low water permeability developed only during storage of tubers in air. Periderm membranes from freshly harvested tubers had a relatively high permeability. The low permeability of PM from stored tubers is attributed to soluble lipids associated with suberin since: (1) extraction of soluble lipids from PM increased permeability by more than 100-fold, (2) a phase transition of soluble lipids was observed between 46 and 51° C, and (3) only the permeability of PM decreased during storage while the permeability of extracted PM remained unchanged. Evidence is presented that two pathways for water movement exist in parallel. Pathway 1 is represented by middle lamellae and primary walls extending in radial direction across the membranes. This pathway has a relatively high specific permeability. Pathway 2 is represented by a polylaminated structure made up of tangential walls of phellem cells which are orientated normal to the direction of water flow. This pathway has a low specific permeability because of the properties of secondary walls incrusted with soluble lipids. It is calculated that about 10% of the water flows across pathway 1 and 90% across pathway 2 which has a volume fraction of 0.995.

Membrane potential oscillations in enzymatically isolated rat myocardial cells.

Enzymatical isolation was used for preparation of single myocardial cells, contractions of which were mainly of slow phasic type but normal fast contractions also occurred. Membrane potential (MP) changes of these cells were measured by conventional microelectrode techniques. Initial MP level of these cells varied between -60 to -90 mV (74.4 +/- 1.9). In the majority of the cells the MP rapidly dropped to -20 to -40 mV and continued to decline while oscillating. In some cells MP recovered from -20 to -40 back to a more negative potential, and the MP oscillation was strongest (up to 20 mV) between -30 to -50 mV. In about 3% of the impalements the MPs did not degenerate at all, but stayed at initial values for several minutes. The MP oscillations were dependent on the MP level and connected to the slow phasic contractions. In connection to the normal fast contractions, two types of action potentials (AP) could be registered. Near -50 mV the cells often generated slow APs of duration 200-400 ms and dV/dt less than 3 V/s but at higher MP level normal fast APs up to 120 mV (100-300 ms and dV/dt greater than 40 V/s) were generated. The slow phasic contractions were never connected to either type of AP.

Characterization of resident glomerular cells in the rat expressing Ia determinants and manifesting genetically restricted interactions with lymphocytes.

The existence of a subpopulation of rat glomerular cells bearing Ia determinants has been demonstrated with the aid of techniques for the enzymatic isolation and culture of glomerular cells. The Ia-positive cell is normally resident in the uninflamed glomerulus. It resembles a mononuclear phagocyte and consists of a functionally heterogeneous cell population with the capacity of Fc receptor display and phagocytosis, both in vivo and in vitro. A new technique for labeling these cells in situ in intact glomeruli has indicated that Ia-positive cells make up approximately 2% of the total glomerular cell population. The isolated glomerular cells can take up antigen and stimulate immune lymphocytes in an I-region-restricted interaction. They are strongly stimulatory in an allogeneic primary mixed lymphocyte culture. Characterization of this cell type suggests potential new insights into the pathogenesis of renal allograft rejection and immunologically mediated glomerulonephritis.

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.

Isolation and some Morphological Properties of Maize Root Protoplasts

Summary A few problems associated with the enzymatic isolation of Zea mays (cv LG 11) root protoplasts were investigated. Beside the importance of the culture conditions, a cysteine pretreatment was shown to give — for different root zones — better digestion of the cell walls and greater stability of the isolated protoplasts. For root zones of increasing differentiation levels, the optimal osmotic pressure of the incubating media increased. Some cytological characteristics of five different protoplast populations, prepared from five distinct zones of 10 mm apical root segments, have been reported.

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

AbstractA method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of 14CO2 fixation both in the light (45 μmol of CO2 fixed mg−1 Chl h−1) and in the dark (20 μmol of CO2 fixed mg−1 Chl h−1). The protoplasts also showed O2 evolution (40 μmol of O2 evolved mg−1 Chl h−1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O2 evolution. Analyses of early photosynthetic products in the light showed the formation of both C3 and C4 acids. Aspartate was found to be a predominant photosynthate.

Procedure for the enzymatic synthesis and isolation of cinnamoyl-CoA thiolesters using a bacterial system

A method is described which allows the preparation of pure cinnamoyl-CoA thiolesters in high yields. This procedure utilizes a partially purified cinnamoyl-CoA ligase obtained from a strain of Pseudomonas putida and some properties of this new enzyme are described. Product isolation involves polyamide column chromatography which allows the purification of 50-mg batches of thiolesters. The method is applicable to a range of cinnamic acids, and is particularly suitable in preparing the biologically important CoA esters of p-coumarate, ferulate, and caffeate.

Fusion of Pea Root Nodule Protoplasts with Tobacco Mesophyll Protoplasts

Summary Protoplasts from pea root nodules were isolated enzymatically. Nodules from flowering pea were suitable material for the isolation of stable protoplasts. The isolated protoplasts were classified into four types ranging from small spheres from the early stage of infected nodule cells (type 1) to large elongated protoplasts from the late stage of infected cells (type 4). In all these types, including the elongated ones, the lack of celL wall materials at the surface of the plasmalemma was confirmed by electron microscopy. The fine structure of vesicles around bacteria and the cytoplasm of the bacteroidal cells appeared well preserved during enzymatic isolation of protoplasts. The isolated nodule protoplasts, especially the spherical types, and tobacco mesophyll protoplasts were agglutinated with polyethylene glycol (PEG), and subsequently fused upon elution of the PEG to form completely spherical fusion products.

Isolement des cellules follaires du soja: activite photosynthetique des suspensions obtenues

A procedure is described for a rapid enzymatic isolation of mesophyll cells from soybean (Glycine max) leaves. A very simple digestion medium (buffer/ osmoticum/enzyme/K+) has been established. The separated cells have rates of light-dependent CO2 fixation of 90 μmol/mg chlorophyll/h; their photosynthetic activity is maintained for up to 4 h. CO2 incorporation is slightly modified when the cells are supplied with exogenous Pi.

Number of nuclei in mammalian cardiac myocytes

The numbers of nuclei per cardiac muscle cell were determined in adult mammalian hearts after previous enzymatic isolation of individual myocytes. A high percentage of binucleation was observed in rat (78 ± 1.8%), rabbit (78 ± 0.8%), guinea pig (81 ± 1.6%), cat (76 ± 1.5%), dog (47 ± 2.4%,), and beef (45 ± 1.1%), while the human myocytes were found predominantly mononucleated (90%) and only 10 ± 2.2% of them were binucleated. These findings are in agreement with our data obtained after serial sectioning of the myocardium of the above species and subsequent reconstruction of individual myocytes. We also found that in fetal and neonatal rat the cardiac myocytes are predominantly mononucleated (95% and 84%, respectively), that the binucleation develops during the early postnatal period, and that adult values are reached by 12–14 days of age. When the body growth and consequently also the heart growth were either accelerated or slowed down by nutritional intervention during the above period, the rate of appearance of binucleation correlated with the organ size rather than with the age of the rat. Early postnatal increase in binucleation was also observed in rabbit, cat, and dog during normal growth. The regional distribution of the above phenomenon was found to be uniform throughout both ventricles.

Aequorin luminescence during activation of single isolated smooth muscle cells.

INCREASED cytoplasmic Ca2+ supposedly couples excitation to contraction in smooth muscle. But there have been no direct measurements of such an increase and there is little information about the kinetics of changes in intracellular Ca2+ during individual contractions of smooth muscle. Thus the basic aspects of excitation–contraction (EC) coupling in smooth muscle are not known with the same degree of certainty that exists for striated muscle1. For example, there is a long delay between stimulation and contraction in smooth muscle; this delay might result from a slow onset of Ca2+ release after a change in surface membrane potential or from the slowness of some other process2. If it were possible to correlate measurements of transmembrane potential, intracellular Ca2+ and contraction in the same single cell the mechanism of EC coupling in smooth muscle might be better understood. Some progress in this direction has been made by the development of methods for enzymatic isolation of smooth muscle cells and measuring electrical activity and force production or shortening in these single cells2–6. We report here a further approach to the problem by (1) microinjecting the Ca2+ indicator aequorin into smooth muscle and (2) directly measuring changes in intracellular Ca2+ during EC coupling in single isolated smooth muscle cells. Cytoplasmic Ca2+ evidently began to rise without detectable delay following a single electrical stimulus applied to a cell injected with aequorin and reached a peak well before contraction presumably began. The long delay, therefore, between stimulation and contraction seems inherent in the mechanism by which the contractile proteins of smooth muscle are activated.