The contribution of alpha1-antitrypsin to the total antitrypsin activity of human serum. A study of two phenotype PI OO individuals.

Comparison of the levels of alpha1-AT, alpha2-M, inter alpha-AT, C1 inactivator and antiplasmin and global antitrypsin activity in a group of normal phenotype PI MM individuals, a group of normal individuals with phenotypes with intermediate alpha1 AT activities and alpha2-AT-deficient persons show that alpha1-AT contributes more than 90 percent of the total antitrypsin activity of normal plasma. AT III and fast reacting antiplasmin are shown to contribute to the remaining activity. It can be assumed that due to test conditions the antitrypsin activity of alpha2-M is not assessed. C1 inactivator and inter alpha1-AT do not contribute to a perceptible extent to the overall antitrypsin activity estimated according to the method of Eriksson (Eriksson, S. (1965) Acta Med. Scand. 177, 1).     

Cystic fibrosis α2-macroglobulin protease interaction in vitro

α₂-Macroglobulin was purified from plasma of five cystic fibrosis patients and five normal controls. SDS gel electrophoresis of native az-macroglobulin from cystic fibrosis patients and normal donors showed identical subunit molecular weights, as did trypsin cleavage products. Cystic fibrosis and control α₂-macroglobulins were indistinguishable by isoelectric focusing and exhibited appropriate shifts in isoelectric point following binding of trypsin. The trypsinbinding capacities of control and cystic fibrosis α₂-macroglobulins did not differ, nor did the esterolytic activity of the trypsin-α₂-macroglobulin complexes.     

Calcium-alginate beads for the oral delivery of transforming growth factor-β1 (TGF-β1): stabilization of TGF-β1 by the addition of polyacrylic acid within acid-treated beads

The susceptibility of the gastrointestinal tract to the toxic effects of chemotherapeutic drugs remains a complication in chemotherapy. Recent studies have suggested that transforming growth factor-β₁ (TGF-β₁) can be used as a cytoprotectant against cell cycle specific drugs. This work describes the use of alginate beads as a potential oral delivery system for TGF-β₁ designed to release the drug in the lumen of the small intestine. TGF-β₁ encapsulation and extent of release from alginate beads approached 100% as determined by ¹²⁵I-labelled TGF-β₁. However, when assayed by ELISA and a growth inhibition assay, nearly all immunoreactivity and bioactivity was lost, apparently due to a very high affinity of the alginate for TGF-β₁. This limitation was overcome by two novel methods: (1) incorporation of selected polyanions within the alginate beads to ‘shield’ TGF-β₁ from interaction with alginate and (2) exposure of the alginate beads containing TGF-β₁ to 0.1 N HCl (acid treatment) to simultaneously reduce the molecular weight of the alginate and its ability to interact with TGF- β₁. If the beads were only acid treated, just 8% of the immunoreactivity ofTGF-β₁ was retained. If polyacrylic acid (90 kDa) was added to the beads, 50% of the immunoreactivity of TGF-β₁ was retained. However, when TGF-β₁ was released from acid-treated beads also containing polyacrylic acid, more than 80% of the TGF-β₁ remained immunoreactive and bioactive. The retained TGF-β₁ activity after release from the beads was found to continue to increase with increasing concentrations of polyacrylic acid, until a concentration was reached where beads would not form. The dramatic increase in retained TGF-β₁ activity is attributed to the ability of polyacrylic acid to shield TGF-β₁ from interaction with lower molecular fragments of alginate.     

HUMAN PLASMA ALPHA 2-MACROGLOBULIN : AN INHIBITOR OF PLASMA KALLIKREIN

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified α₂-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the α₂-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the α₂-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, α₂-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the α₂-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The α₂-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the α₂-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The α₂-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the α₂-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.     

RELATION OF A β1-GLYCOPROTEIN OF HUMAN SERUM TO THE COMPLEMENT SYSTEM

The protein of human serum, tentatively designated β_1C-globulin, was shown to possess serological activity and to be related to the complement system. Another serum protein (β_1A-globulin) was identified as the inactivated form of β_1C-globulin. Incubation of fresh serum with various immune precipitates or with soluble γ-globulin aggregates at 37°C. resulted in the removal of β_1C-globulin. Treatment of fresh serum with zymosan at 17 and 37°C. had a similar effect. In both instances β_1C-globulin was removed from serum, apparently by conversion to β_1A-globulin. However, isolated β_1C-globulin did not react with immune precipitates or zymosan, nor did β_1C-globulin of serum previously heated at 56°C. Highly purified β_1C-globulin was tested for complement component activity by means of the usual reagents. All of the preparations examined were found to reconstitute the hemolytic activity of guinea pig R₃. However, they failed to reconstitute R₃ obtained from human serum. Isolated β_1A-globulin was found to be inactive in all systems. When isolated β_1C-globulin in either phosphate or in borate buffer was stored at 37°C., the activity detected by means of guinea pig R₃ declined within 6 days to 20 to 30 per cent of its original value. As the activity decreased, β_1C-globulin was gradually converted to β_1A-globulin. Addition of β_1C-globulin to a limited complement system (human C') caused an increase of both initial velocity and final degree of hemolysis. Although β_1C-globulin did not cause lysis of EAC'_{1, 4, 2}, it fully prevented the otherwise rapid decay of EAC'_{1, 4, 2} at 37°C., and so presumably interacted with this complex.     

Development of γG, γA, γM, β1C/β1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin, and prealbumin in the human conceptus

The synthesis of γG, γA, γM, β_1C/β_1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, α₁-antitrypsin, orosomucoid, β-lipoprotein, α₂-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in ¹⁴C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized β_1C/β_1A, C′1 esterase inhibitor, transferrin, hemopexin, α₁-antitrypsin, β-lipoprotein, α₂-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of γM occurred as early as 10.5 wk gestation, and γG synthesis was found in cultures as early as 12 wk gestation; γA synthesis was not detected in any of the tissue cultures. With the exception of the γ-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of γG and γM occurred primarily in the spleen, but other sites of synthesis were noted as well.     

Conformational state and receptor recognition of the C-terminal domain of human α2-macroglobulin after dissociation into half-molecules

Background: Dissociation of native human α₂-macroglobulin (α₂M) by sodium thiocyanate generates stable half-molecules with intact thiol esters. Significant conformational changes occur by the dissociation, which are similar to those occurring by transformation from native to methylamine-treated α₂-macroglobulin. Methods: The conformational state of the receptor-binding domain of the half-molecules was investigated by receptor binding and clearance studies, and by use of a panel of 11 monoclonal antibodies (mAbs) specific for the 18-kDa C-terminal receptor-binding fragment of α₂-macroglobulin. Results: The half-molecules simultaneously express epitopes specific for native, as well as epitopes specific for transformed α₂-macroglobulin. While it is possible to immunochemically discriminate between the different forms of tetrameric protein, the half-molecules retain a conformational state with no observed conformational changes in the C-terminal domain following cleavage of thiol esters or bait regions. The in vivo clearance rate in mice was consequently significantly slower for the half-molecules than for the tetrameric receptor-recognized forms of α₂-macroglobulin. Furthermore, half-molecules demonstrate lower affinity for binding to mouse macrophages than methylamine-treated tetrameric α₂-macroglobulin in competition studies. Conclusions: It is suggested that contact zones are functionally important for mediating conformational switches, which result in trapping and exposure of the receptor-binding sites.     

Application of a GM1 ganglioside β-galactosidase microassay method to diagnosis of GM1 gangliosidosis

The enzymatic diagnosis of G_M1 gangliosidosis, including the diagnosis of heterozygosity, requires a microassay of G_M1 ganglioside β-galactosidase activity in lymphocytes and cultured skin fibroblasts. We have adopted high-performance liquid chromatography (HPLC) to the assay of this enzyme and can measure the activity in crude samples fluorometrically. Reaction conditions were examined to determine those optimal for the assay of G_M1 ganglioside β-galactosidase activity in lymphocyte and skin fibroblast homogenates. Under these optimal conditions, reduced enzymatic activities could be detected in lymphocytes and cultured skin fibroblasts from three patients with G_M1 gangliosidosis. Thus, this assay can be used for the diagnosis, rather than the usual assays employing radioactive or artificial substrates.     

Elastase binding capacity of α2-macroglobulin and its association with glucocorticoid concentration in southern African black patients with hepatocellular carcinoma

Thirty-three Southern African black patients with hepatocellular carcinoma (HCC) (7 women) and 43 black control individuals (14 women), all in the age group 18–45 years, were investigated for plasma α₂-macroglobulin (α₂M) elastase binding capacity (EBC). Cortisol levels were measured in 15 (3 women) of the HCC patients and 10 (5 women) of the control subjects. A significant difference in EBC was found between the HCC patients and the control subjects (P < 0.001). A significant difference was also found in cortisol levels between the two groups (P < 0.001). A significant correlation between EBC and cortisol levels was obtained (r = 0.57; P < 0.042). The significant increase in EBC of α₂M in HCC patients could be due to an increase in circulating cortisol.     

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: Effects of prostaglandin F2α

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F₂α (PGF₂α) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF₂α attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF₂α synthesis and PGF₂α is a key stimulator of MMP-2 production. Our data showed that PGF₂α treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF₂α is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.     

Immunochemiluminometric assays (ICMA) specific for growth hormone releasing hormone 1–44 NH2 and 1–40 OH

We describe specific two-site immunochemiluminometric assays able to directly measure human growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH concentrations in unextracted plasma. A common N-terminal antibody was purified from polyclonal rabbit antisera to growth hormone-releasing hormone 1–44 NH₂ on a growth hormone-releasing hormone 1–29 NH₂ linked affinity column and labelled with chemiluminescent acridinium ester. C-terminal specific monoclonal antibodies to growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH were raised in Balb/C mice and used as solid phase antibodies. Assay of fasting specimens from normal individuals gave medians (and ranges) of 23 pg/ml (2–200) and 30 pg/ml (3–134) for growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH, respectively. Samples from a series of acromegalics showed that most have values in the normal range though median values were higher, 56 pg/ml for growth hormonereleasing hormone 1–44 NH₂ (P < 0.001) and 52 pg/ml for 1–40 OH (P < 0.001). Using these assays it will be possible for the first time to directly study the physiology and pathophysiology of these two peptides.     

Diagnosis of Tay-Sachs disease using [H]N-acetylneuraminic acid labelled GM2 ganglioside as substrate

G_M2 ganglioside labelled with tritium in the N-acetylneuraminic acid moiety was prepared and used to measure β-hexosaminidase A activity in cultured human skin fibroblast extracts. The latter convert this substrate to the correspondingly labelled G_M3 ganglioside which can easily be separated from the substrate by thin-layer chromatography. No cleavage of the N-acetylneuraminic acid group was observed under our conditions.Two methods are described for the determination of G_M2-β-hexosaminidase A activity in fibroblasts. The application of these methods to the diagnosis of Tay-Sachs disease is discussed.     

ISOLATION AND CHARACTERIZATION OF TWO β1-GLYCOPROTEINS OF HUMAN SERUM

Two immunoelectrophoretically defined, heretofore unidentified β₁-globulins of human serum, provisionally designated β_1C- and β_1A-globulin, were isolated by means of preparative electrophoresis and chromatography on anion exchange cellulose. The sedimentation coefficient S⁰_{20, w} of β_1C-globulin was shown to be 9.5 S, and that of β_1A-globulin, 6.9 S. Both proteins were found to contain similar amounts of carbohydrate, to be devoid of lipids, and to possess the solubility characteristics of euglobulins. In the Ouchterlony double diffusion test they gave the reaction of partial identity, which revealed β_1A-globulin to be anti-genically deficient as compared to β_1C-globulin. β_1A-globulin could not be detected in fresh sera and β_1C-globulin was absent from aged sera. Highly purified β_1C-globulin stored at 1°C. was converted to β_1A-globulin within 4 to 6 weeks, and at 37°C. was converted within 6 days. The likelihood of a dimer-monomer relationship between these two proteins was discussed.     

Controllable enzymatic superactivity of α-chymotrypsin activated by the electrostatic interaction with cationic gemini surfactants

Surfactant plays a critical role in enzymatic multi-functionalization processes. However, a deep understanding of surfactant-enzyme interactions has been lacking up until now due to the extreme complexity of the mixed system. This work reported the effect of cationic gemini surfactants, alkanediyl-α,ω-bis(dimethyldodecylammonium bromide) (C₁₂C_SC₁₂Br₂, S = 2, 6, and 10) on the enzymatic activity and conformation of α-chymotrypsin (α-CT) in phosphate buffer solution (PBS, pH 7.3). The enzymatic activity was assessed by the rate of 2-naphthyl acetate (2-NA) hydrolysis measured by UV-vis absorption. The superactivity of α-CT in the presence of C₁₂C_SC₁₂Br₂ appears in the concentration region below the critical micelle concentration (cmc) of the surfactant, and its maximum superactivity is correlated to the spacer length of C₁₂C_SC₁₂Br₂. Subtle regulation of the charge density of headgroups of the cationic surfactant can be achieved through partial charge neutralization of cationic headgroups by introducing inorganic counterions or oppositely charged surfactant, demonstrating that the electrostatic interaction plays the crucial role for emergence of the superactivity. The interaction between C₁₂C_SC₁₂Br₂ (S = 2,6, and 10) and α-CT was characterized by isothermal titration calorimetry (ITC), and the obtained endothermic enthalpy change indicates that the interaction induces the change in conformation and enzymatic superactivity. The methodologies of fluorescence spectroscopy, circular dichroism (CD), and differential scanning calorimetry (DSC) show that the gemini surfactants with different spacer lengths induct and regulate the secondary, tertiary and even fourth structures of the protein. The present work is significant to get deeper insight into the mechanism of the activation and denaturation of enzymes.

This study proposes a novel strategy to enhance enzymatic superactivity of α-CT by controlling the charge density of cationic gemini surfactant.     

Enhanced electrochemical performance of α-MoO3/graphene nanocomposites prepared by an in situ microwave irradiation technique for energy storage applications

Nanoparticles of α-molybdenum oxide (α-MoO₃) are directly grown on graphene sheets using a surfactant-free facile one step ultrafast in situ microwave irradiation method. The prepared α-MoO₃ and α-MoO₃/G nanocomposites are analysed by different characterization techniques to study their structural, morphological and optical properties. Transmission electron microscope images reveal the intercalation of three dimensional (3D) α-MoO₃ nanoparticles into 2D graphene sheets without any agglomeration. The electrochemical results exhibit improved performance for the α-MoO₃/G composite electrode compared to pristine α-MoO₃ owing to its structural superiority. The specific capacitance (C_s) values of the α-MoO₃/G composite and pristine α-MoO₃ are measured to be 483 and 142 F g⁻¹ respectively at a current density of 1 A g⁻¹. The α-MoO₃/G composite maintains a very strong cyclic performance after 5000 cycles. The capacitance retention of the composite electrode shows stable behavior without any degradation confirming its suitability as an enduring electrode material for high-performance supercapacitor applications.

Nanoparticles of α-molybdenum oxide (α-MoO₃) are directly grown on graphene sheets using a surfactant-free facile one step ultrafast in situ microwave irradiation method.     

β-N-Acetylhexosaminadases in human cerebrospinal fluid and serum of patients with multiple sclerosis

β-N-Acetylhexosaminidase activity and isoenzyme have been investigated in normal human cerebrospinal fluid and that of patients with multiple sclerosis. β-N-acetylhexosaminidase activity in normal cerebrospinal fluids has been resolved into five components. The major component was in a form that eluted from DEAE cellulose at the same salt concentration as hexosaminidase As, the isoenzyme previously identified in human serum. Cerebrospinal fluid from patients exhibited a different isoenzyme profile, showing a remarkable increase in a form having a pI which was more acidic than that of As. These changes have a potential use in the diagnosis and further biochemical characterization of multiple sclerosis.     

Rationally tailored redox ability of Sn/γ-Al2O3 with Ag for enhancing the selective catalytic reduction of NOx with propene

The development of excellent selective catalytic reduction (SCR) catalysts with hydrocarbons for lean-burn diesel engines is of great significance, and a range of novel catalysts loaded with Sn and Ag were studied in this work. It was found that the synergistic effects of Sn and Ag enabled the 1Sn5Ag/γ-Al₂O₃ (1 wt% Sn and 5wt% Ag) to exhibit superior C₃H₆-SCR performance. The de-NO_x efficiency was maintained above 80% between 336 and 448 °C. The characterization results showed that the presence of AgCl crystallites in the 1Sn5Ag/γ-Al₂O₃ catalyst helped its redox ability maintain an appropriate level, which suppressed the over-oxidation of C₃H₆. Besides, the number of surface adsorbed oxygen (O_α) and hydroxyl groups (O_γ) were enriched, and their reactivity was greatly enhanced due to the coexistence of Ag and Sn. The ratio of Ag⁰/Ag⁺ was increased to 3.68 due to the electron transfer effects, much higher than that of Ag/γ-Al₂O₃ (2.15). Lewis acid sites dominated the C₃H₆-SCR reaction over the 1Sn5Ag/γ-Al₂O₃ catalyst. The synergistic effects of Sn and Ag facilitated the formation of intermediates such as acetates, enolic species, and nitrates, and inhibited the deep oxidation of C₃H₆ into CO₂, and the C₃H₆-SCR mechanism was carefully proposed.

HC-SCR is a more attractive approach for removing NO_x from lean-burn diesel engines compared with NH₃-SCR. The novel SnAg/γ-Al₂O₃ catalyst exhibit excellent C₃H₆-SCR activity, due to the optimized redox ability and enriched hydroxyl groups.     

Complexes between proteinase 3, α1-antitrypsin and proteinase 3 anti-neutrophil cytoplasm autoantibodies: a comparison between α1-antitrypsin PiZ allele carriers and non-carriers with Wegener's granulomatosis

To test the hypothesis that anti-neutrophil cytoplasm autoantibodies (ANCAs) interfere with the functions of proteinase 3 (PR3) (the Wegener autoantigen) and α₁-antitrypsin (α₁AT), complexes of PR3/α₁AT and PR3/PR3-ANCA-IgG were assayed. Plasma samples were obtained from 44 patients with Wegener's granulomatosis (WG): 34 had active disease (88% ANCA positive) whereas 10 patients were in remission (20% ANCA positive). Plasma samples from 14 of the patients with active disease were also available at the time of remission. The complexes of PR3/α₁AT and PR3/PR3-ANCA-IgG were detected by capture enzyme-linked immunoassays (ELISAs). α₁AT deficiency was evaluated by determining PiZ alleles by ELISA. Eight (18%) of the patients were PiZ positive. The frequency of this α₁-antitrypsin phenotype in the Scandinavian population is 4.7% (P < 0.001). The median PR3/α₁AT complex level in the PiZ-positive group with active disease (n = 5) was similar to the level in the PiZ-negative group with active disease. During remission the median level for the PR3/α₁AT complex was significantly higher than in the acute group (P < 0.001) including both PiZ-positive and PiZ-negative patients. No difference between PiZ positivity and PiZ negativity could be found in the remission group. PR3/PR3-ANCA-IgG complexes were found in patients with acute disease as well as in patients in remission, in almost equal frequency. This complex was also present in 13/18 ANCA-negative samples from patients in remission. Finally, purified IgG fractions from WG patients were examined for their capacity to inhibit binding between PR3 and α₁AT. An effect on the binding between PR3 and α₁AT by PR3-ANCA could not be demonstrated. Thus, our results do not support the hypothesis that PR3-ANCA interferes with the binding between PR3 and α₁AT. However, the high prevalence of the PiZ alleles among WG patients suggests that an imbalance between proteinases and α₁AT may be of importance in this disease. The clinical usefulness of both the PR3/α₁AT and the PR3/PR3-ANCA-IgG complexes and the possible influence on ANCA detection need to be examined in prospective longitudinal studies.     

ISOLATION OF β1F-GLOBULIN FROM HUMAN SERUM AND ITS CHARACTERIZATION AS THE FIFTH COMPONENT OF COMPLEMENT

At least 3 complement factors were found necessary for the conversion of the thermolabile intermediate complex EAC'1a,4,2a to a thermostable state. One of these factors is the earlier described β_1C-globulin. The second, a heretofore unrecorded serum protein, β_1F-globulin. The third factor has not yet been defined as a discrete serum protein entity. Kinetic experiments indicated that β_1C reacted prior to β_1F, which in turn seemed to precede the third factor in the reaction sequence. Therefore, the 3 components were tentatively designated the third (C'3), the fifth (C'5), and the sixth (C'6) components of complement, respectively. A procedure was developed allowing the isolation of highly purified β_1C-(C'3) and β_1F-globulin (C'5) and of partially purified C'6. With respect to its function in immune hemolysis, β_1F-globulin or C'5 was found to be closely dependent on the simultaneous presence of C'6. The hypothesis that C'5 and C'6 form a functional unit was supported by the finding that both components interact with each other in solution resulting in the formation of a complex. A similar complex was also found in fresh human serum.     

The β3 subunit of the Na,K-ATPase mediates variable nociceptive sensitivity in the formalin test

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the β₃ subunit of the Na⁺,K⁺-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, β₃ protein expression, and biophysical properties of the Na⁺,K⁺ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the β₃ subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the β₃ subunit of the Na⁺,K⁺-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.