Fluorescence Intensity Of FITC Research Articles

Identification of Novel Ways Disrupting Platelet-Fibrinogen Interactions

Background: Platelets play a central role in thrombosis and hemostasis and immunity through their interaction with fibrinogen. Fibrinogen is a plasma protein that facilitates thrombosis and inflammation. Fibrinogen is the ligand for the major platelet receptor aIIbb3 and TREM- Like Transcript-1 (TLT-1), which platelets release from their a-granules, upon platelet activation. TLT-1 binding fibrinogen was surprising since αIIbβ3, the most abundant platelet receptor, binds fibrinogen, facilitating platelet aggregation. Although TLT-1 may assist in clot formation and hemostasis with αIIbβ3, TLT-1's main association is with regulating inflammatory-derived bleeding. Little is known about the TLT-1-fibrinogen interaction. The goal of this study is to identify the binding sites for this interaction and determine whether it can regulate inflammatory hemostasis in an LPS inflammatory mouse model. Aims: Delineate the TLT-1-fibrinogen binding site and determine if, peptides representing the molecular interaction affect platelet function. Methods: Using an Octet Qke Bio-layer Interferometry (BLI), we confirmed a binding interaction between TLT-1 and fibrinogen. Subsequently we digested fibrinogen using either trypsin or chymotrypsin and immunoprecipitated the peptides with a TLT-1-fc chimera followed by -Mass Spectrometry (LC-MS/MS) to identify potential interacting peptides. As an alternative method, we used a peptide microarray, where fluorescently labeled TLT-1 was incubated with 8-mers of α, β, γ chain fibrinogen peptides attached to a chip to determine which peptides bound TLT-1. Additionally, competitive ELISAs using peptides representing the TLT-1 extracellular domain loops (CDR-1, CDR2, CDR3 and LP17) were incubated with soluble TLT-1 in fibrinogen coated wells to determine the region of TLT-1 responsible for binding fibrinogen. To determine whether any of the selected peptides had inhibitory activity, we used two in vitro competitive inhibitory assays, first, we quantified FITC-labelled fibrinogen binding to activated human platelets and second, human platelet aggregometry incubated in the presence of these peptides at different concentrations (varying from 4-40 μM) using different agonists (ADP, thrombin, collagen, thromboxane A2). Results: We obtained an equilibrium dissociation constant (KD) of 3.02 ± 0.20 nM for the TLT-1 Fibrinogen interaction, suggesting a high affinity interaction. We identified four peptides that had consistent interaction without nonspecific binding in all three trials (trypsin and chymotrypsin IP, as well as the peptide microarray). The competitive platelet aggregometry and flow cytometry assays show decreased aggregation and significantly decreased fluorescent intensity of FITC-labelled fibrinogen in the presence of these peptides, suggesting two of these peptides are our potential binding sites (decrease of FITC-fluorescence intensity when compared to control: p1=20% decrease, p2=44% decrease, p3=21% decrease and P4=39% decrease, p=0.023). A HEK-293 transfected cell line expressing TLT-1 YFP is currently being used to access the ability of the identified peptides to mediate adhesion. In competitive ELISAs, TLT-1 loops LP17 and CDR3 show decreased absorbance when TLT-1 is incubated with fibrinogen in the presence of these peptides. Conclusions: Our TLT-1 data suggests that fibrinogen interacts with TLT-1 in the CD3 loop and in the region designated as LP17. With the aggregometry and flowcytometry competitive assays we have identified p2 as our most promising candidate for the TLT-1 interaction sight on fibrinogen. Currently we are adding the isolated peptides to our HEK/TLT-1 binding assay to determine if our selected peptide yields the same level of inhibition as seen in our aggregation and flow cytometric assays. We will then use an inflammatory in vivo mouse model to determine the effects of administering the selected peptide on platelet neutrophil conjugate formation and mortality. Our poster will report the current state of these studies .

Peptide modified manganese-doped iron oxide nanoparticles as a sensitive fluorescence nanosensor for non-invasive detection of trypsin activity in vitro and in vivo.

Herein, a fluorescence turn-on nanosensor (MnIO@pep-FITC) has been proposed for detecting trypsin activity in vitro and in vivo through covalently immobilizing an FITC modified peptide substrate of trypsin (pep-FITC) on manganese-doped iron oxide nanoparticle (MnIO NP) surfaces via a polyethylene glycol (PEG) crosslinker. The conjugation of pep-FITC with MnIO NPs results in the quenching of FITC fluorescence. After trypsin cleavage, the FITC moiety is released from the MnIO NP surface, leading to a remarkable recovery of FITC fluorescence signal. Under the optimum experimental conditions, the recovery ratio of FITC fluorescence intensity is linearly dependent on the trypsin concentration in the range of 2 to 100 ng mL−1 in buffer and intracellular trypsin in the lysate of 5 × 102 to 1 × 104 HCT116 cells per mL, respectively. The detection limit of trypsin is 0.6 ng mL−1 in buffer or 359 cells per mL HCT116 cell lysate. The MnIO@pep-FITC is successfully employed to noninvasively monitor trypsin activity in the ultrasmall (ca. 4.9 mm3 in volume) BALB/c nude mouse-bearing HCT116 tumor by in vivo fluorescence imaging with external magnetic field assistance, demonstrating that it has excellent practicability.

Silver decahedral nanoparticles-enhanced fluorescence resonance energy transfer sensor for specific cell imaging.

We report on a silver decahedral nanoparticles (Ag10NPs)-based FRET (fluorescence resonance energy transfer) sensor for target cell imaging. Fluorophores-functionalized aptamers (Sgc8-FITC) were bound with Ag10NPs via the SH group on the aptamer to form Ag10-Sgc8-FITC. Then, quencher-carrying strands (BHQ-1) were hybridized with Sgc8-FITC to form a Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q). The sensor interacted with membrane protein tyrosine kinase-7 (PTK-7) on the CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. It was found that Ag10NPs largely enhanced the fluorescence intensity of FITC. The results also showed that the Ag10NPs-based FRET sensor (Ag10-Sgc8-F/Q) was not only superior to the bare FRET sensor (Sgc8-F/Q) and sensor Ag-Sgc8-F/Q but also highly sensitive and specific for CCRF-CEM cells imaging.

Highly sensitive fluorescence detection of copper ion based on its catalytic oxidation to cysteine indicated by fluorescein isothiocyanate functionalized gold nanoparticles

An innovative fluorescence method for sensitive detection of copper ion (Cu2+) was developed based on fluorescein isothiocyanate functionalized gold nanoparticles (FITC-AuNPs). Due to the stronger binding affinity of isothiocyanate functional group to gold, FITC molecules could adsorb on the surface of AuNPs, forming a simple fluorescence resonance energy transfer (FRET) system, and the fluorescence intensity of FITC was remarkably quenched. Upon adding cysteine, FITC could be displaced from the surface of AuNPs because the formation constant (K-f) of Au-S linkage (K-f(AuS-) = 4 x 10(35)) was much higher than AuSCN linkage (K-f(Au(SCN)(2-)) = 10(23)), leading to the recovery of fluorescence intensity. However, Cu2+ could catalyze O-2 oxidation of cysteine, and the generated disulfide cystine could not remove FITC from AuNPs' surface. Therefore, the recovery of fluorescence intensity was much weaker when compared with that of in the absence of Cu2+. And on the basis of this principle the concentration of Cu2+ could be detected quantitatively. Under optimal conditions, our method exhibited high selectivity toward Cu2+ and provided a good linear relationship in the range of 1.0-17.0 nM with the detection limit of 0.37 nM calculated by 3 sigma/S. Furthermore, complicated synthetic procedures and poor water solubility could be ignored in this proposed fluorescent sensor. (C) 2015 Elsevier B. V. All rights reserved.

Recognition Kinetics of Biomolecules at the Surface of Different-Sized Spheres

Bead-based assay is widely used in many bioanalytical applications involving the attachment of proteins and other biomolecules to the surface. For further understanding of the formation of a sphere-biomolecule complex and easily optimizing the use of spheres in targeted biological applications, it is necessary to know the kinetics of the binding reaction at sphere/solution interface. In our presented work, a simple fluorescence analysis method was employed to measure the kinetics for the binding of biotin to sphere surface-bound FITC-SA, based on the fact that the fluorescence intensity of FITC was proportionally enhanced by increasing the binding amount of biotin. By monitoring the time-dependent changes of FITC fluorescence, it was found that the binding rate constant of biotin to sphere surface-immobilized FITC-SA was much smaller than that of biotin to freely diffusing FITC-SA. This can be attributed to the decreased encounter frequency of the reaction pair, restricted motion of the attached biomolecule, and the weakened steric accessibility of the binding site. These factors would become more obvious when increasing the size of the sphere upon which the FITC-SA was immobilized. Additionally, the effect of nanoparticles on the diffusion-controlled bimolecular binding reaction was more evident than that on the chemical recognition-controlled binding reaction.

In-Vitro and In-Vivo Imaging of Prostate Tumor Using NaYF4: Yb, Er Up-Converting Nanoparticles

The aim of this study was to investigate the feasibility of prostate tumor bioimaging both in vitro and in vivo using an upconversion fluorophore, NaYF4: Yb, Er nanoparticles. Luminescent signals of human prostate cancer cells (CWR22R and LNCaP) labeled with NaYF4: Yb, Er nanoparticles were detected by laser scanning confocal microscope, while Cy3 or FITC was used as control probe. Mouse-human prostate cancer model was developed by subcutaneously injecting the CWR22R cells into BALB/c nude mice to investigate the in-vivo imaging properties of NaYF4:Yb, Er nanoparticles. Both CWR22R and LNCaP cells could phagocytose NaYF4:Yb, Er nanoparticles in vitro, and the cellular uptake of CWR22R cells was much higher than that of LNCaP cells (95.42 ± 3.47 % vs. 51.63 ± 6.43 %), which made us choose the former for the further study. CWR22R cells pre-labeled with NaYF4:Yb, Er nanoparticles showed no obvious decrease of fluorescence intensity (P > 0.05) after light exposure, while the fluorescence intensity of Cy3 or FITC labeled cells decreased rapidly with prolonged bleaching (P < 0.05). Furthermore, the in-vivo results showed that the prostate cancer cells pre-labeled with or without NaYF4:Yb, Er nanoparticles formed tumors 4 weeks after injection, and the tumor length-diameter of the nanoparticle group and the control group was (10.3 ± 2.0) mm and (9.8 ± 2.5) mm, respectively. Significant upconversion fluorescence signals were observed in the tumors of the nanoparticle group when being excited at 980 nm by a NIR laser. In summary, the results suggest that as an intensive fluorescence imaging label agent, NaYF4:Yb, Er nanoparticles possess unique features and can be used for imaging prostate tumor cells both in vitro and in vivo by phagocytosis.

Abstract 2620: Progressive expression of n-cadherin and epidermal growth factor receptor in colorectal adenocarcinoma.

Abstract Purpose: N-cadherin (NCAD) and epidermal growth factor receptor (EGFR) have been found to be inducers of epithelial-to-mesenchymal transition that are known to play a role in invasion and metastasis of cancer. Over expression of NCAD and EGFR are associated with increased tumor invasiveness. However, their progressive expression in colorectal cancer (CRC) has not been studied. Therefore, in the present study, we investigated expression status of NCAD and EGFR in paired lesions of CRC. Experimental Design: We examined the expression of NCAD and EGFR in paired lesions of human CRC (hCRC) and human mice CRC (hmCRC) xenograft by immunofluorescence (IF). Images were acquired using the Olympus FluoView FV1000 (OFVFV1000) Confocal Microscope. The average FITC fluorescence intensity (aFFI) was measured by OFVFV1000 software. Western blot analysis was performed to validate results obtained from IF method. Results: Differential expression pattern of NCAD was observed in paired normal, primary and metastatic lesions of hCRC and hmCRC xenograft tissues. For NCAD, it was mainly localized in cytoplasm (diffused), nucleus (moderate levels), and membrane or cell junctions of CRC. For EGFR, normal colon tissue or primary CRC showing membranous EGFR-low positive staining. In contrast corresponding liver metastasis showing membranous EGFR-high positive staining. The NCAD and EGFR expressions were correlated with metastasis phenotype of hCRC. The expressions of NCAD ((aFFI; 812 vs. 261) and EGFR (aFFI 421 vs.131) were each found to be 3 times higher in liver metastasis of hCRC as compared to primary tumor. It has been observed that the expression of NCAD was substantially increased in metastatic lesion of hmCRC xenograft as compared to primary lesion of hmCRC xenograft. Conclusion: This study revealed that upregulation of NCAD and EGFR expressions is a mechanism by which CRC lesions acquire a more aggressive phenotype and, thereby, progress to a more advanced stage of CRC. Furthermore, our findings are likely to have implications in the design of clinical trials targeting the NCAD and EGFR proteins in CRC. Citation Format: Venkat R. Katkoori, Upender Manne, Marc D. Basson, Harvey L. Bumpers. Progressive expression of n-cadherin and epidermal growth factor receptor in colorectal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2620. doi:10.1158/1538-7445.AM2013-2620

Fluorescent Polysaccharide Nanoparticles for pH-Sensing

The exploration of chemical microenvironments in organisms as well as on the cellular level is of great interest in medical and biological research. Therefore, dextran nanoparticles, which are labelled with both a pH-indicator dye (fluorescein isothiocyanate, FITC) and a reference dye (sulforhodamine B acid chloride) as an internal standard, were developed as biocompatible nanosensores that can be used for ratiometric pH measurements. The ratio of the fluorescence dyes can easily be tuned during the nanoparticle formation by choosing the appropriate mixture of the labelled derivatives prior the nanoprecipitation. The fluorescence intensity of FITC increases with increasing pH value, whereas the signal of the reference dye remains constant. The polysaccharide based pH-nanosensors are characterized in terms of fluorescence lifetime, autoclaving stability, response to ionic strength, oxidation and photobleaching. The resulting pKa of 6.45 is appropriate for most of the measurement purposes. Fluorescence lifetime measurements indicate that energy transfer between the dyes takes places. However, no negative influence on the performance of the pH value measurements could be observed.

Evaluation of fluorescent polysaccharide nanoparticles for pH-sensing

The comprehensive characterization of novel dextran nanoparticles with regard to their suitability as pH-sensors for analytical applications (e.g. in physiology) is described. The nanoparticles are labeled with both a pH-indicator dye (fluorescein isothiocyanate, FITC) and a reference dye (sulforhodamine B acid chloride) as an internal standard. The fluorescence intensity of FITC increases with increasing pH, whereas the signal of the reference dye remains constant. Plotting the ratio of both signals against the pH gives a pK(a) of 6.45, which is appropriate for most of the measurement purposes. Furthermore, the influence of temperature, ionic strength and oxidizing substances on the performance of the fluorophores inside the dextran nanoparticles is examined. These results are compared to the dissolved dyes in order to evaluate if the dextran matrix affects the fluorescence properties of the sensor and the reference dye, and whether or not these nanosensors are suitable for pH-sensing in biological samples.

Rational Design of Intercellular Adhesion Molecule-1 (ICAM-1) Variants for Antagonizing Integrin Lymphocyte Function-associated Antigen-1-dependent Adhesion

The interaction between integrin lymphocyte function-associated antigen-1 (LFA-1) and its ligand intercellular adhesion molecule-1 (ICAM-1) is critical in immunological and inflammatory reactions but, like other adhesive interactions, is of low affinity. Here, multiple rational design methods were used to engineer ICAM-1 mutants with enhanced affinity for LFA-1. Five amino acid substitutions 1) enhance the hydrophobicity and packing of residues surrounding Glu-34 of ICAM-1, which coordinates to a Mg2+ in the LFA-1 I domain, and 2) alter associations at the edges of the binding interface. The affinity of the most improved ICAM-1 mutant for intermediate- and high-affinity LFA-1 I domains was increased by 19-fold and 22-fold, respectively, relative to wild type. Moreover, potency was similarly enhanced for inhibition of LFA-1-dependent ligand binding and cell adhesion. Thus, rational design can be used to engineer novel adhesion molecules with high monomeric affinity; furthermore, the ICAM-1 mutant holds promise for targeting LFA-1-ICAM-1 interaction for biological studies and therapeutic purposes.

Catalytic Activity of ADAM8, ADAM15, and MDC-L (ADAM28) on Synthetic Peptide Substrates and in Ectodomain Cleavage of CD23

The ADAM family of disintegrin metalloproteases plays important roles in "ectodomain shedding," the process by which biologically active, soluble forms of cytokines, growth factors, and their receptors are released from membrane-bound precursors. Whereas ADAM8, ADAM15, and MDC-L (ADAM28) are expressed in specific cell types and tissues, their in vivo functions and substrates are not known. By screening a library of synthetic peptides as potential substrates, we show that soluble recombinant forms of these enzymes have similar proteolytic substrate specificity, clearly distinct from that of ADAM17 (TNFalpha-converting enzyme). A number of tumor necrosis factor (TNF) family proteins and CD23 were screened as potential substrates for ectodomain cleavage. We found that ADAM8, ADAM15, and MDC-L, but not ADAM17, catalyzed ectodomain shedding of CD23, the low affinity IgE receptor. ADAM8-dependent, soluble CD23 release required proteolytically active ADAM8, and a physical association of ADAM8 was observed with the membrane-bound form of CD23. The ADAM8-dependent release of sCD23 and the endogenous release from B cell lines could be similarly inhibited by a hydroxamic acid, metalloprotease inhibitor compound. We conclude that ADAM8 could contribute to ectodomain shedding of CD23 and may thus be a potential target for therapeutic intervention in allergy and inflammation.

A Novel Multiparametric Approach for Analysis of Cytoplasmic Immunoglobulin Light Chains by Flow Cytometry

We describe a novel flow cytometric approach using a two-step acquisition technique to determine the cytoplasmic immunoglobulin light chains (LC) expression. Samples were prepared by a lysed-whole-blood technique and incubated with CD38-PE (phycoerythrin) and CD45-FITC (fluorescein isothiocyanate). The cells were fixed and acquired on an FACSCalibur flow cytometer (first acquisition). The cells were then permeabilized, incubated with either kappa-FITC or lambda-FITC and reacquired (second acquisition). Analysis of the data was performed by gating on the differing intensities of CD38 and evaluating them for the presence of a shifting FITC-positive population from the first acquisition to the second acquisition. The FITC fluorescence intensity of the second acquisition was equal to the sum of surface CD45 expression obtained during the first acquisition and the cytoplasmic LC expression obtained during the second acquisition. Thus, the shifting (increase) of FITC fluorescence intensity during the second acquisition is specifically due to the cytoplasmic expression of either the kappa or lambda LC. We studied 15 multiple myeloma (MM) patients and 10 controls (samples from patients without plasma cell dyscrasias). None of the controls showed evidence of any clonal populations. Thirteen of 15 MM patients exhibited clonal plasma cells (CD38 bright), ranging from 0.01% to 34% of total events collected. In addition, we identified another minute clonal population of lymphocytes (CD38 dim, CD45 bright, low forward and side scatter) in 12 of 13 MM patients with clonal plasma cells. This population, ranging from 0.01% to 0.6% of total events collected, had the same LC restriction as the clonal plasma cells. Patients with a ratio of minor clonal population to clonal plasma cells less than 0.07 tended to remain in partial or complete remission than those with a ratio ≥0.07 (4/5 versus 1/4, P < .1, χ2). We conclude that this method is highly sensitive and permits us to identify the minute clonal population of lymphocytes in MM patients. Our preliminary observations with a small cohort of patients imply that this minute clonal population may have important prognostic significance. The prognostic significance should be confirmed by further studies.

ICAM‐1‐expressing pocket epithelium, LFA‐1‐expressing T cells in gingival tissue and gingival crevicular fluid as features characterizing inflammatory cell invasion and exudation in adult periodontitis

Activated T lymphocytes constitute a major component of inflammatory cells in the early periodontal lesion, and also appear in the gingival crevicular fluid. In an attempt to clarify the relationship between the ICAM-1 (CD54) expression of pocket epithelium in gingiva and the infiltrating lymphocyte population, we carried out an analysis of CD11a+(LFA-1 alpha), CD25+(IL-2R alpha) and CD4+(Th) cells subjacent to ICAM-1-expressing pocket epithelia and CD11a+CD25+CD4+ cells in gingival crevicular fluid (GCF). GCF was collected by crevicular washing from 16 patients with periodontitis (P group) and 3 subjects with healthy gingiva (H group). Peripheral blood (PB) was collected at the same time. Mononuclear cells were isolated by Ficoll-paque gradient centrifugation from GCF and PB. Monoclonal antibodies (mAb) to CD11a, CD25, and CD4 were used for three-color flow cytometry. Gingival biopsies were obtained from 7 patients in P group and 3 subjects in H group. Serial cryostat sections (6 microns in thickness) were prepared from each biopsy, on which a double staining was performed. The number of CD11a+CD25+CD4+ cells and the fluorescence intensity of FITC conjugated anti-CD11a were significantly higher in GCF than in PB (p < 0.001 to p < 0.01). CD11a+CD25+CD4+ cells were not detected in GCF in H group. The pocket epithelia expressed CD54 in P group, but not in H group. The number of CD11a+, CD25+ and CD4+ cells infiltrating the connective tissue subjacent to the upper, middle and lower parts of the CD54 positive pocket epithelium (n = 16) was 141 +/- 26, 38 +/- 13, 144 +/- 29 (cells/0.04 mm2), respectively, whereas in the CD54 negative pocket epithelium, it was (n = 5) 9 +/- 2, 3 +/- 1, 8 +/- 3. In P group, the CD11a+CD25+CD4+ cell number in GCF correlated with CD25+, CD11a+ cells in the connective tissue subjacent to the CD54+ pocket epithelium. These results indicate that expression of ICAM-1 in pocket epithelium is relevant to the migration of CD11a, CD25, CD4 positive cells in connective tissue subjacent to the pocket epithelium into the periodontal pocket. Assessing the relationship of our findings and other adhesion molecules would offer important clues to the understanding of T cell migration in affected gingiva.

Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could be discriminated from unlabelled mitoses, and from labelled and unlabelled G2 cells, by their intermediate log FITC fluorescence intensity. In addition, mitoses and G2 nuclei differed in forward and orthogonal light scattering, but had equal intensity of propidium iodide fluorescence. This method for discrimination of labelled mitoses was also tested on cultured normal adult human keratinocytes labelled with iododeoxyuridine (IdUrd). In keratinocytes, where the cell structure was preserved after pepsin/HCl, IdUrd labelled mitotic cells were similarly discriminated in the log FITC/propidium iodide fluorescence distribution. This interpretation was supported by experiments using mitotic arrest, fluorescence activated cell sorting and microscopy, and comparison with an alternative flow cytometric method for discrimination of mitoses.

Fiber-optic biosensors based on fluorescence energy transfer

A new optical homogeneous biochemical method for the assay of glucose has been developed, based on fluorescence energy transfer between a glucose analog, dextran labeled with fluorescein isothiocyanate (FITC—dextran), and a glucose-receptor protein, Rhodamine-labeled Concanavalin A (Rh—ConA). When FITC—dextran binds to Rh—ConA in solution, and is light-activated, the FITC label transfers its absorbed energy to the Rhodamine label, which then emits light according to its own characteristic fluorescence spectrum. When glucose is added to this solution, the FITC fluorescence intensity increases as FITC—dextran is released from the Rh—ConA and is replaced by glucose. Thus it is possible to determine glucose concentrations directly from the level of FITC fluorescence.

Immunochemical evidence that the FITC-labeling site on Na +,K +-ATPase is not the ATP binding site

The covalent labeling of the α subunit of lamb kidney Na +,K +-ATPase by fluorescein 5′-isothiocyanate at Lys-501 has generally been assumed to occur at the ATP binding site. We have found that the peptide sequence 496HLLVMKGAPER 506 serves as the antigenic determinant for monoclonal antibody M8-P1-A3. This antibody binds to both native and FITC-labeled enzyme and while this epitope undergoes ligand-induced changes these changes are not involved in either enzyme function or the E 1 ⇄ E 2 conformational changes monitored by FITC-fluorescence intensity.

Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase.

The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O-methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p-nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high-affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the (-K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the (-K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high-affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1).