Fish Viruses: Buffers and Methods for Plaquing Eight Agents Under Normal Atmosphere

A universal procedure was sought for plaque assay of eight fish viruses (bluegill myxovirus, channel catfish virus, eel virus, Egtved virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, lymphocystis virus, and the agent of spring viremia of carp (Rhabdovirus carpio), in dish cultures of various fish cells. Eagle minimal essential medium with sodium bicarbonate-CO(2) buffer (Earle's salt solution) was compared with minimal essential medium buffered principally with tris (hydroxymethyl)aminomethane or N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid at a pH or in the range of 7.6 to 8.0 depending upon temperature. Five fish cell lines collectively capable of replicating all fish viruses thus far isolated were tested and quantitatively found to grow comparably well in the three media. Two-phase (gel-liquid) media incorporating the various buffer systems allowed plaquing at 15 to 33 C either in partial pressures of CO(2) or in normal atmosphere, but greater efficiency and sensitivity were obtained with the organic buffers, and, overall, the best results were obtained with tris(hydroxymethyl)aminomethane. Epizootiological data, specific fish cell line response, and plaque morphology permit presumptive identification of most of the agents. At proper pH, use of organic buffers obviates the need for CO(2) incubators.     

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.     

The role of low intracellular or extracellular pH in sensitization to hyperthermia

Cells are more sensitive to heat when they are heated in an acidic environment, and this study confirms (K. G. Hofer and N. F. Mivechi, J. Natl. Cancer Inst., 65, 621, 1980) that intracellular pH (pHi) and not extracellular pH (pHe) is responsible for the sensitization. The relationship between pHe, pHi, and heat survival of cells heated in vitro in various buffers at pHe 6.3-8.0 was investigated. Cells' adaptation to low environmental pH in terms of increases in pHi and heat survival also was investigated. Finally, we studied the relationships among pHe, pHi, and survival from heat for cells heated in sodium-free reconstructed medium. Intracellular pH was measured by the distribution of the weak acid, [2-14C]5,5-dimethyl-2,4-oxazolidinedione. Our results are summarized as follows: (1) CHO cells maintained the same relationship between pHe and pHi in four different media or buffers (McCoy's 5a medium buffered with CO2 and NaHCO3 or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) and 2-(N-morpholino)ethanesulfonic acid (Mes), Krebs-Ringer bicarbonate solution, and Krebs-Ringer phosphate solution) with pHi being 0.05 to 0.20 pH units higher than pHe as it varied from 7.0 to 6.4; furthermore, heat sensitization by acid was the same in medium buffered with NaHCO3 or Hepes and Mes. (2) The low pHe adapted cells multiplied with an increased doubling time of 20.7 +/- 0.7 h and appeared morphologically similar to the unadapted cells. However, the pHi of these cells was 0.15-0.30 pH units higher than that of the unadapted cells when pHe was varied between 7.0 and 6.3. (3) After being heated at 43.5 degrees C for 55 min or at 42.5 degrees C for 150 min at pHe 6.3-7.2, the pHi of the adapted cells increased by 0.2-0.1 pH units. However, heat caused no significant change in the unadapted cells. (4) Heat survival plotted versus pHe was 1000-fold higher for the adapted cells than for the unadapted cells at pHe of 6.3. However, heat survival plotted versus pHi was identical for the two cell types. (5) In sodium-free reconstructed McCoy's 5a medium, pHi was 0.25-0.1 pH units lower than that in the sodium-containing counterpart at pHe 6.3-7.2, and heat sensitization increased accordingly; however, heat survival plotted versus pHi was identical for the two types of media.     

The effect of pH on the population growth of three species of duckweed: Spirodela oligorrhiza, Lemna minor and Wolffia arrhiza

Three species of duckweed, Spirodela oligorrhiza, Lemna minor and Wolffia arrhiza were grown under aseptic conditions on both buffered and unbuffered solutions of Jacob's media. Media with manually regulated pH levels were also used. Growth on unbuffered media is initially rapid but eventually inhibited, probably by increased pH levels. On buffered media growth is poor and effects of buffers cannot be separated out. These media give inadequate pictures of the species’ responses to changes in pH. Growth is most successful on media with regulated pH where sustained logarithmic population increases were achieved. Spirodela and Lemna rates are symmetrical about an almost neutral, optimal pH, declining fairly rapidly away from the optimum. Wolffia has an optimum at pH 5 and growth declined with increasing pH. All three species have optima at, or below, the neutral point. The range of tolerance of duckweeds is broader than has previously been suspected. Estimated lower limits, optimum and upper limits for each species are: Wolffia, pH 4·5–0·10, Lemna pH 4–6·2–10, Spirodela pH 3·7–0·10. Growth rate along a pH gradient is best described by means of polynomial equations: second-degree equations are sufficient for Spirodela and Lemna but a fifth-degree equation is required for Wolffia. Rates of population growth are similar for all species. In decreasing order they are: Wolffia, Lemna, Spirodela. However, in biomass units Lemna grew more than six and Spirodela seventeen times faster than Wolffia.     

Roles of calcium and pH in activation of eggs of the medaka fish, Oryzias latipes

Unfertilized eggs of the medaka fish (Oryzias latipes) were injected with pH-buffered calcium buffers. Medaka egg activation is accompanied by a transient increase in cytoplasmic free calcium (Gilkey, J. C., L. F. Jaffe, E. B. Ridgway, and G. T. Reynolds, 1978, J. Cell Biol., 76:448-466). The calcium buffer injections demonstrated that (a) the threshold free calcium required to elicit the calcium transient and activate the egg is between 1.7 and 5.1 microM at pH 7.0, well below the 30 microM reached during the transient, and (b) buffers which hold free calcium below threshold prevent activation of the buffered region in subsequently fertilized eggs. Therefore an increase in free calcium is necessary and sufficient to elicit the calcium transient, and the calcium transient is necessary to activate the egg. Further, these results are additional proof that the calcium transient is initiated and propagated through the cytoplasm by a mechanism of calcium- stimulated calcium release. Finally, a normal calcium transient must propagate through the entire cytoplasm to ensure normal development. Unfertilized eggs were injected with pH buffers to produce short-term, localized changes in cytoplasmic pH. The eggs were then fertilized at various times after injection. In other experiments, unfertilized and fertilized eggs were exposed to media containing either NH4Cl or CO2 to produce longer term, global changes in cytoplasmic pH. These treatments neither activated the eggs nor interfered with the normal development of fertilized eggs, suggesting that even if a natural change in cytoplasmic pH is induced by activation, it has no role in medaka egg development. The injected pH buffers altered the rate of propagation of the calcium transient through the cytoplasm, suggesting that the threshold free calcium required to trigger calcium-stimulated calcium release might be pH dependent. The results of injection of pH-buffered calcium buffers support this conjecture: for a tenfold increase in hydrogen ion concentration, free calcium must also be raised tenfold to elicit the calcium transient.     

pH sensitivity of H+/organic cation antiport system in rat renal brush-border membranes

We examined the pH sensitivity of the H+/organic cation antiport system in brush-border membranes isolated from rat renal cortex. The uptake of tetraethylammonium, a typical organic cation, in the absence of an H+ gradient had a marked pH dependence with an optimum pH of 7.0, while the uptake of p-aminohippurate, an organic anion, and D-glucose was almost consistent in the pH range of 6.0-8.0. The decreased tetraethylammonium uptake by brush-border membrane vesicles, suspended in an acidic pH buffer or an alkaline pH buffer, was completely recovered by subsequent treatment of the vesicles with a pH 7.0 buffer. The pH sensitivity of tetraethylammonium uptake was not changed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a protonophore, or valinomycin (voltage-clamped condition). Kinetic parameters of tetraethylammonium uptake were changed in a pH-dependent manner, although Eadie-Hofstee plots of tetraethylammonium uptake were linear in the pH range of 6.0-8.0, indicating the existence of one mode of transport system at various pH values. At an acidic pH, the Km was increased without any change in Vmax value, compared with the values at pH 7.0. On the other hand, at an alkaline pH, the Vmax was decreased without a change in Km value. These results suggest that the H+/organic cation antiport system in renal brush-border membranes is very sensitive to pH (optimum pH of 7.0), in contrast to organic anion and D-glucose transport systems, and that pH is an important factor to regulate the activity of the H+/organic cation antiport system, as well as H+ gradient (a driving force).     

Ion chromatographic quantification of cyanate in urea solutions: estimation of the efficiency of cyanate scavengers for use in recombinant protein manufacturing

The chaotrope urea is commonly used during recombinant protein manufacturing as a denaturant/solublizing agent. The adventitious accumulation of cyanate in urea solutions during product manufacturing can cause unwanted carbamylation of proteins, leading to alterations in drug product structure, stability and function. We have developed an ion chromatographic method to quantify cyanate production in urea solutions, suitable for analysis of samples from manufacturing process buffers. We discuss assay development, system suitability criteria and limitations on assay applicability. The assay has a linear range from 2 to 250 microM, with LOQ/LOD values of 6 and 2 microM, respectively. Assay accuracy through spike/recovery testing were established and both precision and intermediate precision were estimated. We assessed the utility of the assay by testing a variety of biological buffers and potential cyanate scavengers, which could be used during protein purification processes, for their ability to control the level of cyanate in 8 M urea solutions buffered over the range of pH 5-10. Our results demonstrate pH dependence for prevention of cyanate accumulation by these buffers/scavengers and indicate useful buffers, pH ranges, and additives for controlling cyanate accumulation during recombinant protein manufacturing. The pertinence of these approaches in preventing protein carbamylation during manufacturing are discussed.     

Assays of invertase activity in acidic soils: Influence of buffers

Summary The influence of buffered and unbuffered systems for assays of invertase activity in a range of acidic soils (pH4.9–6.8), and a neutral soil (pH 7.1), from under pasture was determined. The buffers were those recently recommended in other studies,viz. a modified universal buffer (MUB) and a potassium phosphate buffer. The optimum pH for the invertase activity of a moderately acid soil (pH 5.5) wasc 4.0 and for the neutral soil was 5.0 With the acidic soils, invertase activity was lower in the assay system with MUB (initial pH 5.0) than in the unbuffered system, and decreased with increasing MUB molarity. The phosphate buffer was more satisfactory, even though the pH (5.0) was below its most effective range. Generally, either phosphate buffer or unbuffered systems appear suitable for measuring invertase activity in these acidic soils.     

Effect of temperature and pH on the ribulose-1,5-diphosphate carboxylase activity of the thermophilic hydrogen bacterium, Pseudomonas thermophila

The activity of ribulose 1,5-diphosphate (RDP) carboxylase was found in the soluble fraction of the cytoplasm from sonicated Pseudomonas thermophila K-2 cells. The enzyme is relatively thermolabile and completely loses its activity at 80 degrees C. The activity of RDP carboxylase at 60 degrees C increases by 40% during the first 10 min of heating in the presence of Mg2+ ions, bicarbonate and dithiothreitol, and again decreases if the enzyme is heated over 20 min. The optimum temperature of the enzyme is 50--55 degrees C. The specific activity of the enzyme in fresh preparations under these conditions reaches 0.22 unit per 1 mg of protein in the extract. The calculated value of the activation energy for RDP carboxylase is 6.4 kcal-mole-1, but 11.6 kcal-mole-1 in frozen preparations. The optimal pH is 7.0--7.3 depending on the buffer. The temperature optimum for the enzyme action does not depend on pH within the range of 7.3 to 8.8. Therefore, RDP carboxylase of Ps. thermophila K-2 differs from RDP carboxylases of mesophilic cultures studied earlier by a higher susceptibility to a decrease in temperature (the enzyme activity is negligible at 30 degrees C), by a lower value of the activation energy at suboptimal temperatures, and by a lower pH optimum of the enzyme action.     

Mechanism of CO2 acquisition in an acid-tolerant Chlamydomonas

The acid-tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using (14)C-benzoic acid (BA) or [2-(14)C]-5,5-dimethyloxazolidine-2,4-dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0-7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO(2) uptake and O(2) evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO(2) uptake. The CO(2) compensation concentration of the cells ranged from 9.4 microM at pH 4.5 to 16.2 microM at pH 7.0. An examination of the kinetics of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the K(m) (CO(2)) was 16.3 microM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO(2), and the expression of external CA at neutral pH values would maintain an equilibrium CO(2) concentration at the cell surface. This species does not possess a CO(2)-concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low K(m) (CO(2)) Rubisco of the alga.     

Proline accumulation induced by weak acids and IAA in coleoptiles of wheat seedlings

Proline accumulation in coleoptiles of wheat seedlings or in excised coleoptile segments incubated under shaking for a 24 h period was studied. There was no increase of proline content of coleoptiles after incubation of the seedlings in 5 mM citric acid (a relatively strong and slowly penetrating organic acid) in a pH range from 4.5 to 7.0 and only a slight increase of proline content after incubation in phosphate buffer at pH 7.0 to 7.5 duo to the higher osmotic concentration of phosphate buffer in this pH range. Quite different results were obtained with seedlings incubated in 10 mM acetic acid, a weak and easily penetrating organic acid. With increasing proton concentrations, proline accumulation increased. Application of 400 mM mannitol or higher concentrations of IAA (more than 10⁻⁵M) additionally increased proline accumulation in the presence of 10 mM acetic acid in the pH range from 6.0 to 7.5 in which acetic acid alone was loss effective. It is suggested that a decrease of cytosolic pH causes stress—induced proline accumulation.     

Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant

To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3- at chemical equilibrium is essentially unaffected by the amino acid substitution at pH greater than 7.0 and slightly decreased in the mutant at pH less than 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by others buffers, such as MOPS or HEPES. Similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N-bis[N, N-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.     

Cytochemical, histological, and phylogenetic distribution of a 38,000-dalton protein associated with transverse tubules

A major protein in detergent extracts of skeletal muscle appears at 38,000 daltons in electrophoretic separations. Previous investigations have provided indirect evidence that a 38-kD skeletal muscle protein is membrane associated, and this inference has served as the basis for speculations on 38-kD protein function. In the present study, affinity purified, polyclonal antisera against 38-kD protein from skeletal muscle are produced for immunolocalization and biochemical assays. Immunoblots of two-dimensional electrophoretic separations show that this protein is heterogenously charged at pI approximately 6.4. This 38-kD protein is not extracted from muscle in low ionic strength or high ionic strength buffers, in isotonic buffers from pH 4 to pH 8 or in buffers containing 5 mM EGTA. The 38-kD protein is extracted, however, by isotonic, pH 7.0 buffer containing 1.0% Triton-X. Light microscope observations using indirect immunofluorescence of anti-38-kD labeled tissue show the protein distributed in a reticular pattern within cross-sectional muscle but not at the cell surface. Longitudinal sections show the protein concentrated in periodic, transverse bands. Purified fractions of muscle plasma membrane analyzed by immunoblotting contain 38-kD protein. Immunoblots using anti-38 kD show that this protein is present in all vertebrate skeletal muscle examined, however, the protein is present only in cardiac muscle that contains transverse tubules. The antibody does not recognize aldolase, another 38-kD striated muscle protein.     

Effect of moderately acidic pH on heat resistance of Clostridium sporogenes spores in phosphate buffer and in buffered pea puree.

The effect of pH in the range 5.0 to 7.0 on the thermal destruction of spores of Clostridium sporogenes putrefactive anaerobe 3679 was examined by three methods: a capillary tube method in which spores were suspended in phosphate buffers, a thermoresistometer method in which spores were suspended in buffered pea puree adjusted to the same set of pH values, and a thermal death time can method in which spores were again suspended in buffered pea puree. The results indicated that increasing acidity is, in general, accompanied by decreasing heat resistance, although the pH effect was more pronounced at the higher than at the lower processing temperatures. Certain pH values appear to be critical, as they produced, in all three sets of experiments, effects which would not be predicted by the overall relationship between acidity and spore heat resistance. Differences between heat resistance in phosphate buffer as compared with that in pea puree adjusted to the same pH were also noted. D-values in buffer were found to be lower than those in pea puree, except at the highest temperatures coupled with the lowest pH values. The differences between buffer D-value and pea puree D-value were found to increase with increasing pH and with decreasing temperature. On the other hand, at all pH values examined, z-values determined in buffer were somewhat higher than those determined in pea puree adjusted to the same pH.     

Effect of decreased pH on force and phosphocreatine in mammalian skeletal muscle

Phosphocreatine (PCr) and intracellular pH changes were monitored by 31P-NMR spectroscopy in isolated, arterially perfused cat biceps and soleus muscles, while the pH of the CO2-bicarbonate buffered perfusate was decreased from 7.1-7.4 to 6.4-6.7 by increasing the CO2 in the equilibrating gas from 5 to up to 70%. In biceps (fast twitch) muscles, intracellular pH decreased from 7.0 to 6.6 (30% CO2, 30 degrees C), peak tetanic force decreased by 8%, but the rise and relaxation times of tetanic were not significantly changed. In soleus muscles, intracellular pH decreased from 7.0 to 6.6 (30% CO2, 30 degrees C), peak tetanic force was unchanged, but the rise and relaxation times of tetani were increased by 27 and 112%, respectively. In both muscles greater decreases in tetanic force were observed during repetitive or ischemic stimulation, which resulted in intracellular pH similar to that produced by hypercapnia. Contrary to previous reports, there was no significant decrease in PCr level in either muscle type with decreased intracellular pH. In the soleus at 30 degrees C there was a significant increase in PCr level with decreased pH.     

High-activity biocatalysts in organic media: solid-state buffers as the immobilisation matrix for protein-coated microcrystals

Recently, we reported a new high-activity biocatalyst for use in organic media termed protein-coated microcrystals (PCMC) (Kreiner et al. [2001] Chem Commun 12:1096-1097). These novel particles consist of water-soluble micron-sized crystalline particles coated with the given biocatalyst(s) and are prepared in a one-step rapid dehydration process. In this study we extended the choice of immobilisation matrix from a simple inorganic salt, K(2)SO(4), to other compounds, both inorganic and zwitterionic, that act as solid-state buffers for biocatalysis in organic media. The catalytic activity of serine proteases subtilisin Carlsberg (SC) and alpha-chymotrypsin (CT) were significantly increased when coated onto the surface of solid-state buffers, as measured in acetonitrile/1wt% H(2)O. SC-PCMC with both organic and inorganic buffer carriers (Na-AMPSO, Na(2)CO(3), and NaHCO(3)) showed a 3-fold greater activity than that observed when using the unbuffered system (PCMC-SC/K(2)SO(4)). In comparison with freeze-dried preparations, this represents an approximately 3,000-fold increase in catalytic activity. Importantly, there is no improvement in catalytic activity upon external addition of any of the solid-state buffers to the reaction mixture. When acting in a solid-state buffer capacity, good buffering capacity was observed with SC-PCMC (3 wt% protein loading) prepared from a 1:1 mixture of AMPSO and AMPSO-Na. Alternatively, increasing the amount of solid-state buffer in the system allows improvement of the buffering. This can be achieved either by decreasing the protein loading of the SC/Na-AMPSO-PCMC or by addition of further external solid-state buffer to the reaction mixture. The catalytic activity of lipase-PCMC prepared from solid-state buffers was found less responsive to immobilisation.     

Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.     

Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.     

An adenosine triphosphate-dependent stabilization of proteolytic activity in heterolysosomes. Evidence for a proton pump

1. Homogenization of mouse kidneys or livers in 0.25m-sucrose buffered with tris-acetate, pH7.3, resulted in a decreased rate of proteolysis within isolated heterolysosomes containing injected (125)I-labelled albumin when these particles were incubated at 35 degrees C. Proteolysis in mouse kidney or liver heterolysosomes isolated from homogenates made in 0.25m-sucrose buffered at pH7.3 was stimulated by pH5 buffer or by additions of ATP. 2. A greater inhibition of proteolysis was produced by including bicarbonate or pH8 borate buffers in the incubation media, and this inhibition was also reversed by ATP. 3. Other nucleoside triphosphates were not as effective as ATP, but GTP and ITP were more effective than CTP or UTP. ADP, AMP, or adenosine 3':5'-cyclic monophosphate were completely without effect. 4. Although ATP prevented some heterolysosome breakage in media containing bicarbonate, the primary effect appeared to be to promote proteolytic activity. 5. These observations are consistent with the presence of a proton pump in the heterolysosome membrane, which functions to maintain intralysosomal pH in alkaline media.