Large Increase In Fluorescence Research Articles

Nutrient control of phytoplanktonic biomass in atoll lagoons and Pacific ocean waters: Studies with factorial enrichment bioassays

Although the atolls of the Tuamotu archipelago (Central South Pacific) are located in an oligotrophic oceanic area, some of their lagoons have experienced exceptionally harmful phytoplanktonic blooms in the last 30 years. Twenty-four differential enrichment bioassays were conducted on 10 atoll lagoons and 5 ocean sites at two different times of the year in order to determine, among other factors, which nutrients may control phytoplanktonic crop. Complete factorial (2 3) design with N, P and Si and fractional factorial (2 8−3) design with N, P, Si, chelator, Fe, Mo, Mn and vitamins were performed. In vivo fluorescence (IVF) was used to follow the growth of phytoplankton. Although this method is imperfect, we argue that the large increases in fluorescence, observed in response to some spikes, indicate biomass shifts. Nitrogen, phosphorus and sometimes silicate effects were significant. The nitrogen effect was greatest in 17 out of 24 samples. In the smallest lagoon, the phosphorus effect was higher than the nitrogen effect. In the six other samples N and P effects were similar. Silicate spikes resulted in a significant effect for only seven samples. Vitamins, Mo, Mn, iron and chelators had little or no effect. In 20 bioassays there was also a synergistic effect when N and P were added simultaneously. This synergistic effect was present in five bioassays when N, P, and Si were added simultaneously. The season or origin (lagoon or ocean) had little influence on these effects. On average fluorescence attained by samples supplemented with N alone was six times that of controls (unspiked). The highest responses were observed with combined N+P or N+P+Si spikes. Combined N+P fertilization produced an in vivo fluorescence ranging from 5 to 85 times the fluorescence attained by controls. This synergy is consistent with the view that both N and P are in relatively short supply. Ocean waters appeared to be nitrogen depleted. Phosphorus limitation increased in small lagoons with low advective ocean waters and a large ratio of immersed surfaces to water volume. Atoll lagoons and ocean waters from the Tuamotu archipelago appear to be highly susceptible to eutrophication with the addition of both nitrogen and phosphorus.

Crystal structure of phosphate binding protein labeled with a coumarin fluorophore, a probe for inorganic phosphate.

Crystal structures are presented for the A197C mutant of Escherichia coli phosphate binding protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Both proteins are complexed with inorganic phosphate. The latter molecule, MDCC-PBP, exhibits a large increase in fluorescence on binding inorganic phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational changes associated with inorganic phosphate binding are interpreted in terms of the specific interactions of MDCC with the protein. The structure helps to explain why this particular label gives a high-fluorescence state on binding inorganic phosphate, while several other related labels do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on proteins.

Detection and Quantitation of Heterotrimeric G Proteins by Fluorescence Resonance Energy Transfer

N-Methyl-3′-O-anthranoyl (mant) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for detection of heterotrimeric guanine nucleotide binding proteins. The mant derivative of GTPγS, mGTPγS, is synthesized and purified by modification of a method initially described by Hiratsuka (1983,Biochim. Biophys. Acta742, 496–508). The binding affinity of mGTPγS for G proteins Giand Gois comparable to that of GTPγS. The rate of binding is determined by the dissociation rate of the endogenously bound GDP. The large fluorescence increase observed upon mGTPγS binding to G protein is due, in part, to resonance energy transfer from tryptophans in the G protein to the mant guanine nucleotide. The magnitude of the fluorescence increase is dependent upon the concentration of G protein. Therefore, mGTPγS binding can be used to quantitate and locate G proteins during the protein purification process. This method is rapid compared to the [35S]GTPγS binding assay in that (i) the bound ligand does not need to be separated from the free ligand thus avoiding vacuum filtration and (ii) the time required to measure fluorescence in each sample is less than that required for scintillation counting. In addition, the use of radioactivity can be avoided. Thus, the mGTPγS binding assay for the detection of Giand Gorepresents a rapid, reliable alternative to assays based on radiolabeled GTPγS binding or ADP-ribosylation with pertussis toxin.

Mitochondrial membrane potential and hydroethidine-monitored superoxide generation in cultured cerebellar granule cells

Mitochondrial depolarisation has been reported to enhance the generation of superoxide anion (O ⋅− 2) in a number of cell preparations while an inhibition has been observed with isolated mitochondria. Cerebellar granule cells equilibrated with >1 μM hydroethidine (dihydroethidium) which is oxidised to the fluorescent ethidium cation by O ⋅− 2 showed a large increase in fluorescence on protonophore addition. However, controls showed the fluorescent enhancement to be a consequence of release of unbound preformed ethidium from the mitochondrial matrix within the cell with resultant fluorescent enhancement. This ambiguity was removed by the use of low (1 μM) concentrations of dye in which case generated ethidium remained bound within the mitochondria. Under these conditions antimycin A, but not protonophore addition, produced an increase in fluorescence. It is concluded that excess ethidium acts as an indicator of mitochondrial membrane potential obscuring the monitoring of O ⋅− 2 and that certain experiments employing this indicator in cells may require re-evaluation.

Catalytic Properties of Lipopolysaccharide (LPS) Binding Protein: TRANSFER OF LPS TO SOLUBLE CD14

Lipopolysaccharide (LPS) binding protein (LBP) is a lipid transfer protein that catalyzes transfer of LPS monomers from micelles to a binding site on soluble CD14 (sCD14) and transfer of LPS from LPS.sCD14 complexes to HDL particles. To characterize the first of these two reactions, LPS covalently derivatized with the fluorophore, boron dipyrromethene difluoride (BODIPY), was used to monitor LBP-catalyzed movement of LPS in real time. The fluorescence efficiency of micelles of BODIPY-LPS was low but was strongly increased upon dissolution in detergent or upon binding to sCD14. Spontaneous binding of BODIPY-LPS to sCD14 was very slow but was accelerated by substoichiometric concentration of LBP, and the rate of binding was measured under a variety of conditions. LBP-catalyzed transfer was first order with respect to both sCD14 and LPS concentration, and the apparent Km values were 1 approximately 2 microg/ml for sCD14 and 100 ng/ml for LPS. The maximum turnover number for LBP was approximately 150 molecules of LPS min-1 LBP-1. LBP alone caused a small but measurable increase in the fluorescence of BODIPY-LPS, suggesting that it bound LPS aggregates but did not readily remove LPS monomers. The subsequent addition of sCD14 caused a large fluorescence increase, suggesting transfer of BODIPY-LPS to sCD14. These and other observations suggest that LPS is transferred by an ordered ternary complex reaction mechanism in which LBP transfers LPS monomer from LPS aggregates to sCD14 without dissociating from the LPS aggregate.

On the interaction of α-crystallin with unfolded proteins

α-Crystallin, a major protein component of the lens, has chaperone-like properties whereby it prevents destabilised proteins from precipitating out of solution. It does so by forming a soluble high-molecular-weight (HMW) complex. A spectroscopic investigation of the HMW complex formed between a variety of unfolded proteins and bovine α-crystallin is presented in this paper. As monitored by fluorescence spectroscopy, a large amount of the hydrophobic probe, 8-anilino-1-naphthalene sulfonate (ANS) binds to the HMW complex implying that the complexed proteins (alcohol dehydrogenase (ADH), γ-crystallin and rhodanese) are bound in an unfolded, possibly molten-globule state. The interaction between the anionic surfactant, sodium dodecyl sulfate (SDS) and ADH at high temperatures gives rise to a similar large increase in ANS fluorescence to that for the complex between α-crystallin and ADH. SDS, like α-crystallin, therefore complexes to proteins in their unfolded state leaving a large hydrophobic surface exposed to solvent. Unlike other chaperones (e.g., GroEL, DnaK and SecB), α-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated α-lactalbumin and α-casein). It is concluded that α-crystallin will only complex with proteins that are about to precipitate out of solution, i.e., ones that are severely compromised. 1H-NMR spectroscopy of the HMW complex formed between α-crystallin and γ-crystallin indicates that the short C-terminal extension of α B-crystallin, but not that of α A-crystallin, has lost its flexibility in the complex implying that the former is involved in interactions with the unfolded γ-crystallin molecule, possibly electrostatically via its two C-terminal lysine residues.

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent "amplification" was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high-NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)

Polyethenoadenosine Phosphate as a Fluorogenic Substrate for Barnase

Polyethenoadenosine phosphate is rapidly hydrolyzed by barnase with an accompanying large increase in fluorescence. At pH 8, 0.2 M ammonium acetate, the initial rate of fluorescence increase is proportional to barnase concentration and thus provides an excellent quantitative assay for its activity. The preparation and use of this substrate in the barnase assay is presented in detail.

Ca2+, caldesmon, and myosin light chain kinase exchange with calmodulin.

Wheat calmodulin (CaM) was labeled at Cys-27 with the sulfhydryl-specific fluorescent probe 2-(4'-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS), to form MIANS.CaM. In the presence of Ca2+, MIANS.CaM undergoes a large fluorescence increase when it binds myosin light chain kinase (MLCK) and caldesmon (CaD), but little fluorescence change when it binds CaM antagonists or Ca2+. MLCK associates with MIANS.CaM at a rate of 2.8 x 10(7) M-1 s-1 and dissociates at 0.031 s-1 (Kd = 1.1 nM). Protein kinase A phosphorylation of MLCK (P-MLCK) produces a 3.5-fold decrease in its association rate with CaM and a 6-fold increase in its dissociation rate (Kd = 23 nM). CaD associates with MIANS.CaM with a rate of 5.3 x 10(8) M-1 s-1 and dissociates at 57 s-1 (Kd = 108 nM). EGTA disrupts the CaM.MLCK, CaM.P-MLCK, and the CaM.CaD complexes at rates of 3.5 s-1, 6.5 s-1, and 13.5 s-1, respectively. MLCK, therefore, dissociates from CaM more quickly by Ca2+ removal while the lower affinity CaD is dissociated more quickly by competition from higher affinity CaM target proteins than by Ca2+ removal. MLCK binding to CaM slowed Ca2+ dissociation from CaM's C-terminal Ca(2+)-binding sites from 30 s-1 to 6 s-1 while CaD had little effect on Ca2+ dissociation from these sites. During a Ca2+ transient, CaM could exchange with MLCK and CaD rapidly enough for these proteins to be directly involved in the contraction/relaxation cycle of smooth muscle.

Blue Light-Modulation of Chlorophyll a Fluorescence Transients in Guard Cell Chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO(2) concentration on the fluorescence transients was investigated in the presence of HCO(3) (-) at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO(2) concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.

An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence

Proteinases and their inhibitors have become the subject of intense research interest recently, since they control a multitude of very important biological processes, from the development of λ phage to hypertension in humans. We have developed a simple and sensitive assay for detecting the activity of proteinases and of their proteinase inhibitors. The assay is based on ethidium bromide fluorescence, according to the following principles: (i) Ethidium bromide increases its fluorescence by 25-fold when it intercalates between base pairs of double-stranded DNA. (ii) Histones prevent this large increase in fluorescence by binding with high affinity to DNA thus blocking ethidium bromide intercalation. (iii) A proteinase that digests histones will make more DNA available for ethidium bromide intercalation, thereby producing an increase of fluorescence. Proteinase activity can easily be determined, in the presence of a DNA/histone complex, from the rate of ethidium fluorescence increase. In contrast, activity of a proteinase inhibitor is quantitated by the inhibition of fluorescence gain in the presence of a known amount of proteinase. This assay is rapid, simple, inexpensive, and, at the same time, accurate and sensitive enough to allow quantitation of nanogram amounts of various broad-specificity proteinases and their inhibitors. We show some possible applications of the assay (i) in testing column fractions during protein purifications, (ii) quantitation of α 1-antitrypsin in human serum, and (iii) detection of proteinase activity in cell extracts.

Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding .alpha.-bungarotoxin

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)

2′,3′-O-(2,4,6-Trinitrophenyl)-8-Azido-adenosine Mono-, Di-, and Triphosphates as Photoaffinity Probes of the Ca2+-ATPase of Sarcoplasmic Reticulum: Regulatory/Superfluorescent Nucleotides Label the Catalytic Site with High Efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence (superfluorescence) during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to fish myosin and the effect of salts on the thermal transitions of fish myosin-ANS complex

Myosin prepared from tilapia ( Serotherodon aureus) was complexed with 8-anilino-1-naphthalene sulfonate (ANS) and continuously heated at 1 °C/min. A large increase in fluorescence was observed with a transition temperature of 34 °C. The effect of several salts on the transition temperature was tested. A plot based on the equation of E. E. Schrier and E. B. Schrier [(1967) J. Phys. Chem. 71, 1851–1860] gave a value of </500 cal/ mol-deg for the change in enthalpy per residue due to exposure to solvent. The ratio of hydrophobic group to amide group exposure to solvent was intermediate compared with the ratio of RNase and gelatin. Fluorescence titrations yielded one high affinity site with a K b of 2 × 10 6 m −1 and at least 200 low affinity sites with an average value of 1 × 10 5 m −1. The parameters did not change significantly with temperature. We propose that the increase in ANS fluorescence reflects changes in conformation of myosin as monitored by these low affinity sites, resulting in an increase in surface hydrophobicity and representing a small enthalpic change in the conformation of the myosin molecule. As a consequence, the change in conformation accelerates polymerization of myosin oligomers.

Heme protein fluorescence versus pressure

Fluorescence spectra of several ferric heme proteins have been measured vs. pressure to 6,000 bars. Sperm whale myoglobin (SW Mb), Aplysia myoglobin, leghemoglobin (Lb), and cytochrome P450 all show excitation and emission spectra characteristic of tryptophan in proteins with peak emission at 330-340 nm. At one bar, the fluorescence is weak due to energy transfer to the heme group, which makes the yield a sensitive probe of protein unfolding at high pressure. After an initial decrease of a few percent per kbar, the protein shows a large increase in fluorescence at high pressure. The increase is pH dependent and the results indicate that several high pressure states occur. For SW Mb at 15 degrees C an increase of a factor of 20 occurs with midpoint at 2,000 bars at pH 5 and is only partially reversible, while the increase at pH 7 occurs at 4,000 bars and is only half as large and is completely reversible. Aplysia Mb and Lb show a similar effect, but unfold at a higher pressure than SW Mb. P450 also shows a transition to a state of higher fluorescence, but the transition in this case is irreversible as a stable form, P420, is formed. The fluorescence intensity measurements permit an estimation of the increase in the TRY-heme distance in the high pressure state.

A fluorescence study of the binding of poly(1, N6-ethenoadenylic acid) to Escherichia coli initiation Factor 3

The binding of initiation Factor 3 (IF3) to poly(1, N 6 -ethenoadenylic acid) [poly(ϵA)] was investigated by fluorescence spectroscopy. At low salt concentrations, IF3 evokes an increase in the fluorescence intensity of poly(ϵA) due to the unstacking of the nucleotide bases. The poly(ϵA) fluorescence enhancement titrates to an endpoint of 13 ± 2 nucleotide residues per IF3. The maximum poly(ϵA) fluorescence enhancement, at lattice saturation, decreases with increasing salt concentration. Even though IF3 does not produce a large fluorescence increase between 75 and 200 m m NaCl concentration, the protein still binds to poly(ϵA) at these salt concentrations as measured by sedimentation partition chromatography; the value of K obs for the IF3-poly(ϵA) interaction is comparable to that of other synthetic polynucleotides. The binding of IF3 to poly(A) at 150 and 200 m m NaCl induces an increase in nucleotide base-base separation as determined by CD, yet IF3-induced disruption of base stacking of poly(ϵA) at these same salt concentrations is not detected by fluorescence. It is likely that IF3 binds primarily to the phosphate backbone of poly(ϵA) at low salt concentrations, producing an increase in the fluorescence intensity. But, at higher salt concentrations, the aromatic amino acids intercalate between the nucleotide bases quenching the poly(ϵA) fluorescence.

Photosynthetic characteristics of phycoerythrin-containing marine Synechococcus spp.

Marine Synechococcus spp. are sufficiently abundant to make a significant contribution to primary productivity in the ocean. They are characterized by containing high cellular levels of phycoerythrin which is highly fluorescent in vivo. We sought (Jan.–Apr., 1984) to determine the adaptive photosynthetic features of two clonal types of Synechococcus spp., and to provide a reliable physiological basis for interpreting remote sensing data in terms of the biomass and productivity of this group in natural assemblages. It was found that the two major clonal types optimize growth and photosynthesis at low photon flux densities by increasing the numbers of photosynthetic units per cell and by decreasing photosynthetic unit size. The cells of clone WH 7803 exhibited dramatic photoinhibition of photosynthesis and reduction in growth rate at high photon flux densities, accompanied by a large and significant increase in phycoerythrin fluorescence. Maximal photosynthesis of cells grown under 10–50 μE m-2 s-1 was reduced by 20 to 30% when the cells were exposed to photon flux densities greater than 150 μE m-2 s-1. However, steady-state levels of photosynthesis maintained for brief periods under these conditions were higher than those of cells grown continuously at high photon flux densities. No photoinhibition occurred in clone WH 8018 and rates of photosynthesis were greater than in WH 7803. Yields of in-vivo phycoerythrin fluorescence under all growth photon flux densities were lower in clone WH 8018 compared to clone WH 7803. Since significant inverse correlations were obtained between phycoerythrin fluorescence and Pmax and μ for both clones grown in laboratory culture, it may be possible to provide a reliable means of assessing the physiological state, photosynthetic capacity and growth rate of Synechococcus spp. in natural assemblages by remote sensing of phycoerythrin fluorescence. Poor correlations between phycoerythrin fluorescene and pigment content indicate that phycoerythrin fluorescence may not accurately estimate Synechococcus spp. biomass based on pigment content alone.

Intracellular calcium storage and release in the human platelet. Chlorotetracycline as a continuous monitor.

The calcium-sensitive fluorescent probe chlorotetracycline was used to monitor calcium movement in human platelets. The chlorotetracycline fluorescence signal is a linear measure of the level of free calcium in the dense tubules and in the mitochondria, with probe sensitivity in the millimolar range. Experiments perturbing the system with the calcium ionophore A23187 shows that the level of free internal calcium in the organelle depends upon the cytoplasmic level, which, in turn, depends upon the passive permeability of the plasma membrane. Chlorotetracycline in the cytoplasmic compartment does not respond to changes in the cytoplasmic calcium concentration, which is held in the micromolar to submicromolar range by an extrusion system. The calcium concentration in the cytoplasmic compartment can be directly manipulated by the calcium ionophore A23187 and is measured in parallel experiments with Quin 2, a high-affinity indicator. The calcium transport systems of the organelles are shown to be less susceptible to short circuit by A23187. Analysis shows that mitochondrial uptake is slow (t 1/2 = 20 minutes), produces a large increase in chlorotetracycline fluorescence, and is inhibited by sodium azide plus oligomycin. Uptake by the dense tubules is more rapid (t 1/2 = 2 minutes), produces a smaller increase in chlorotetracycline fluorescence, is inhibited by trifluoperazine, and is less sensitive to A23187. The Km is estimated as 1 microM or lower. Studies show that the chlorotetracycline technique is useful for the monitoring of calcium uptake and release by the platelet organelles, and suggests that the Quin 2/chlorotetracycline technique will be useful as a diagnostic of both physiological and pathological activation mechanisms.

Lipoprotein lipase- and phospholipase A 2-catalyzed hydrolysis of phospholipid vesicles with an encapsulated fluorescent dye effects of apolipoproteins

The self-quenching dye, 6-carboxyfluorescein, has been encapsulated into sonicated vesicles of egg phosphatidylcholine. Porcine pancreatic phospholipase A 2 and bovine milk lipoprotein lipase catalyze the hydrolysis of the phosphatidylcholine resulting in the release of the encapsulated dye and a large increase in 6-carboxyfluorescein fluorescence. The fluorescence increase occurs in parallel with the formation of lysophosphatidylcholine and is strongly dependent on Ca 2+ for phospholipase A 2 catalysis and on apolipoprotein C-II for hydrolysis by lipoprotein lipase. Other apolipoproteins, including apolipoproteins C-III, C-I, and A-I, do not enhance lipoprotein lipase activity towards this substrate. We conclude that the enhancement of lipoprotein lipase activity by apolipoprotein C-II is a specific property of the activator protein due to its interaction with lipoprotein lipase or an enzyme/lipid interface and not a characteristic of lipid-binding proteins in general.

Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge

Upon mixing detergent solutions with the neutral fluorescent molecule 1,6-diphenyl-1,3,5-hexatriene a large increase in fluorescence is observed if detergent exceeds the critical micelle concentration. This property has been used to determine the critical micelle concentration of anionic, uncharged, zwitterionic, and cationic detergents. Regardless of detergent charge, the critical micelle concentrations obtained agree with the values obtained by other methods. This fluorescence assay is both sensitive and rapid, and should provide a simple and general method for determination of critical micelle concentration of any detergent.