Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection

A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 degrees C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5'-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5+/-40.9 micromol/l (n=79), with cerebral vascular disorders 12.3+/-7.0 micromol/l (n=65), and with coronary atherosclerosis 15.4+/-10.9 micromol/l (n=15) than that in healthy subjects (6.2+/-1.74 micromol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.     

Interactions of cadmium and selenium in rat plasma in vivo and in vitro.

75Se and 109Cd tracers were used to study the binding of Se and Cd to plasma proteins at various SeO32- doses and times upt to 24 h after the simultaneous subcutaneous administration of SeO32- markedly increased both Se and Cd plasma levels over that in control animals. Gel permeation chromatography of plasma indicated that at all times up to 24 h Cd and Se were bound in an atomic ratio of approx. 1 : 1 in 330 000 and 130 000 dalton fractions. From 4 to 24 h, Cd and Se appeared in the 420 000 dalton fraction, also with an atomic ratio of approx. 1 : 1. The 330 000 dalton molecules appeared to have a maximal binding capacity for the Cd-Se complex at a concentration of approx. 30 mumol/ml of plasma, while the 130 000 and 420 000 dalton molecules show a higher binding capacity. Studies in vitro revealed that SeO32- does not interact directly with Cd and plasma proteins. It is metabolized by erythrocytes to a form that interacts in an atomic ratio of 1 : 1 with Cd to form a protein-bound complex of 130 000 daltons.     

Plasma protein binding of APD: role of calcium and transferrin.

The bisphosphonate drug APD (pamidronate, 3-amino-1-hydroxypropylidene-1,1-bisphosphonate) has been shown to bind to human plasma proteins. This was an unexpected observation since this hydrophilic, anionic drug is not typical of molecules that exhibit this characteristic. At a concentration of 5 micrograms/ml the extent of binding of APD to fresh human plasma in vitro was variable between subjects 30.2% +/- 8.5% (mean +/- S.D., n = 10). Binding was not influenced by the time or concentration of APD over the range 0.05-10.0 micrograms/ml. At 20 and 50 micrograms/ml some precipitation of APD occurred. Both calcium and iron play a role in the binding of APD to plasma proteins, addition of calcium to plasma increased the degree of binding of APD, whereas the calcium chelators EDTA and EGTA reduced the binding of APD. Similarly, addition of iron to plasma increased the binding and the inclusion of the iron chelator desferrioxamine diminished the binding of the drug. The effects of iron and desferrioxamine were less pronounced than those of calcium and EDTA, indicating that the majority of the binding involves calcium ions and a smaller contribution is made by ferric ions. The equilibrium dissociation constants (Kd) for APD binding to calcium and iron binding sites on plasma proteins were estimated to be 852 microM and 29 microM, respectively. Calcium binding sites were of high capacity but low affinity and the iron binding sites were of lower capacity and higher affinity. Electrophoresis of plasma proteins following incubation with [14C]APD revealed binding to the transferrin and globulin fractions. However, there was some dissociation of protein bound APD during the electrophoresis. The consequences of hypercalcaemia on the pharmacokinetics of APD are discussed.     

Uptake and/or binding of tricyclic antidepressants in human red cells

The factors affecting the red blood cells (RBC) uptake and/or binding of tricyclic antidepressants imipramine (IMI) and desmethylimipramine (DMI) were investigated under in vitro conditions. The time course of drug distribution indicated that equilibrium between RBC and medium is reached rapidly. The uptake and/or binding of IMI and DMI to RBC was not saturable and RBC/medium ratio was unaffected by pH, nature of monovalent cations or by the presence of therapeutic concentrations (5-50 ng/ml) of antipsychotic drugs. In the protein-free medium, RBC/medium ratio of DMI was X +/- SE = 2.58 +/- 0.08 (n = 5) and that of IMI was 7.38 +/- 0.12 (n = 6). However, RBC/plasma ratio of DMI was 0.78 +/- 0.04 (n = 7) and IMI ratio was 0.64 +/- 0.03 (n = 9). The RBC/medium ratio was linearly related to free fraction of IMI in a buffer containing 60 mg/ml of human serum albumin with various concentrations of alpha 1-acid glycoprotein (r = 0.971, n = 7). These results suggest that in the absence of proteins the lipid solubility of antidepressants is the main determinant factor in RBC uptake and/or binding, whereas, in the presence of proteins, free fraction of the drug plays the major role. Hence, the RBC/plasma ratio of antidepressants may correlate with treatment outcome in clinical studies better than total drug concentration in plasma.     

Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells

Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions.     

Heat inhibition of in vitro lipolysis and 14C ibuprofen protein binding in plasma from heparinized uraemic subjects

Protein binding determination in post heparin plasma samples is complicated by the continued post heparin lipase activity, in vitro, during the binding analysis. The decomposition of lipoproteins and accumulation of nonesterified fatty acids (NEFA) results in artifically elevated free fractions of many drugs. This artefact is particularly accentuated in haemodialysis patients who are frequently hypertriglyceridaemic and receive large doses of heparin. Rapid heat treatment (60 degrees for 15 min) of plasma from heparinized uraemic subjects is shown to inhibit the in vitro lipolysis occurring during 2 hours of equilibrium dialysis at 37 degrees (ED). Mean NEFA concentrations in heat treated plasma after ED (means = 400 +/- 141 mumol/L) were not different (p greater than 0.05, n = 9) from the baseline values in fresh plasma (means 351 +/- 117 mumol/L) but were considerably less (p less than 0.005) than NEFA levels in untreated plasma after ED (means = 1025 +/- 523 mumol/L). The degree of in vitro lipolysis inhibition (92 +/- 6.6%) was very much greater than using the chemical inhibitors phenyl methyl sulphonyl fluoride, EDTA, Triton X100 or protamine sulphate. Heat treatment at 60 degrees for 15 min increased the percentage of free 14C ibuprofen in 3.5% isolated human serum albumin from 0.34% to 0.62%. Reduced binding as a result of heat treatment was not observed however in whole plasma. The percentage free ibuprofen in heat treated, whole plasma from both heparinized and non heparinized subjects (means = 1.22 +/- 0.19; n = 29) was not different (p greater than 0.05) from the percentage free determined in plasma from a non heparinized group (means = 1.16 +/- 0.23; n = 15). In contrast the % free ibuprofen in untreated plasma from heparinized subjects was markedly higher (means = 1.56 +/- 0.41; n = 24; p less than 0.05). There was a strong correlation between % free ibuprofen and plasma NEFA concentration (r = 0.8; p less than 0.005; n = 68). The heat treatment of plasma for 15 min at 60 degrees is proposed as an effective means of controlling heparin induced lipolysis in vitro and may be valuable in overcoming the post heparin binding artefact.     

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.     

Sex steroid binding proteins in the plasma of hatchling <Emphasis Type="Italic">Chelonia mydas</Emphasis>

Sex steroid binding proteins were identified in hatchling female and male Chelonia mydas by dialysis and steady-state gel electrophoresis when examined at 4 degrees C. A testosterone binding protein with high binding affinity (K (a) = 0.98 +/- 0.5 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 7.58 +/- 4.2 x 10(-5) M) was observed in male hatchlings. An oestradiol binding protein with high affinity (K (a) = 0.35 +/- 1.8 x 10(8) M(-1)) and low to moderate binding capacity (B (max) = 0.16 +/- 0.5 x 10(-4) M) was identified in female hatchlings. This study confirmed that sex steroid binding proteins (SSBPs) become inactivate in both sexes at 36 degrees C, the maximum body temperature of sea turtle hatchlings at emergence. The inactivation of SSBPs at this temperature indicates that sex steroid hormones circulate freely in the body of the green turtles and are biologically available in the blood plasma. This observation is consistent with female and male hatchling C. mydas having different physiological (hormonal) and developmental requirements around the time of emergence. Moreover, concurrently conducted competition studies showed that sex steroids including testosterone and oestradiol do compete for binding sites in both male and female C. mydas hatchling plasma. Competition also occurred between testosterone and dihydrotestosterone for binding sites in the male C. mydas plasma. However, competition studies in the plasma of female hatchling C. mydas demonstrate that oestrone does not compete with oestradiol for binding sites.     

Plasma albumin cysteinylation is regulated by cystathionine beta-synthase

High homocysteine (Hcy) levels are a well-known independent risk factor for endothelial damage in atherosclerosis. We examined whether a rat intestinal model of ischemia-reperfusion was associated with high Hcy and with the modification of plasma albumin into cysteinylated species (CysAlb). The three treatment groups were as follows: midline abdominal incision (group A, n=10), followed by ligation of the superior mesenteric artery for a period of 2h (group B, n=3), and followed by reperfusion for 1h (group C, n=10). Hcy levels were 2.5-fold higher in group C than group A (p<0.05). 100% and 73.44+/-0.04% of Alb were modified into Cys species in groups C and B, respectively, compared to 51.2% in group A. A cystathionine beta-synthase (CBS) deficient mouse model, known to have high plasma Hcy levels, was also used to determine the extent of CysAlb. Hcy levels, %CysAlb, and %HcyAlb were 180.1+/-45.7 microM, 0%, and 23.4+/-4.4% in CBS deficient mice, while in control mice, those values were 5.7+/-1.8 microM, 24.2+/-4.1%, and 0%, respectively (p<0.05). High CysAlb and Hcy levels were observed in a rat model of bowel ischemia/reperfusion while high HcyAlb and Hcy levels with no CysAlb were observed in the CBS deficient mice. CysAlb may serve as a biomarker for the severity of gut ischemia, and high Hcy may explain endothelial damage associated with this model. Additionally, active CBS is essential for the formation of CysAlb.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

Differences in plasma homocysteine levels between Zucker fatty and Zucker diabetic fatty rats following 3 weeks oral administration of organic vanadium compounds

PURPOSE: Recently, our laboratory group has reported that rats with Type 1 diabetes have decreased plasma homocysteine and cysteine levels compared to non-diabetic controls and that organic vanadium treatment increased plasma homocysteine concentrations to non-diabetic concentrations. However, to date, no studies have been done investigating the effects of organic vanadium compounds on plasma homocysteine and its metabolites in Type 2 diabetic animal model. These studies examined the effect of organic vanadium compounds [bis(maltolato)oxovanadium(IV) and bis(ethylmaltolato)oxovanadium(IV); BMOV and BEOV] administered orally on plasma concentrations of homocysteine and its metabolites (cysteine and cysteinylglycine) in lean, Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rats. ZF rats are a model of pre-diabetic Type 2 diabetes characterized by hyperinsulinemia and normoglycemia. The ZDF rat is a model of Type 2 diabetes characterized by relative hypoinsulinemia and hyperglycemia. METHODS: Zucker lean and ZF rats received BMOV in the drinking water at a dose of 0.19 +/- 0.02 mmol/kg/day. Lean and ZDF rats received BEOV by oral gavage daily at dose of 0.1 mmol/kg. The treatment period for both studies was 21 days. At termination, animals were fasted overnight (approximately 16 h) and blood samples were collected by cardiac puncture for determination of plasma glucose, insulin and homocysteine levels. Plasma homocysteine and its metabolites levels were determined using high-pressure liquid chromatography. Plasma glucose was determined using a Glucose Analyzer 2. Plasma insulin levels were determined by radioimmunoassay. Plasma triglycerides were determined by an enzymatic assay methodology. RESULTS: ZF (n = 4) and ZDF (n = 10) rats had significantly lower plasma homocysteine as compared to their respective lean groups (ZF 0.78 +/- 0.1 micromol/L vs. Zucker lean 2.19 +/- 0.7 micromol/L; ZDF 1.71 +/- 0.2 micromol/L vs. Zucker lean 3.02 +/- 0.3 micromol/L; p < 0.05). BMOV treatment in ZF rats restored plasma homocysteine levels to those observed in lean untreated rats (ZF treated: 2.04 +/- 0.2 micromol/L; lean 2.19 +/- 0.7 micromol/L). There was a modest effect of BMOV treatment on plasma glucose levels in ZF rats. BEOV treatment significantly decreased the elevated plasma glucose levels in the ZDF rats (lean 7.9 +/- 0.1 mmol/L; lean + vanadium 7.7 +/- 0.2 mmol/L; ZDF 29.9 +/- 0.4 mmol/L; ZDF + vanadium 17.4 +/- 0.3 mmol/L, p < 0.05). Organic vanadium treatment reduced cysteine levels in both ZF and ZDF rats. No differences in total plasma cysteinylglycine concentrations were observed. CONCLUSION: Plasma homocysteine levels are significantly reduced in a pre-diabetic model of Type 2 diabetes, which was restored to lean levels upon vanadium treatment; however, this restoration of plasma homocysteine levels was not seen in ZDF Type 2 diabetic rats following vanadium treatment. In the latter case vanadium treatment may not have totally overcome the insulin resistance seen in these animals.     

Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Pharmacokinetics of sulfadimethoxine in cats

Pharmacokinetic parameters which describe distribution and elimination of sulfadimethoxine were determined in cats. Following intravenous administration of a single dose (55 mg/kg), disposition of the drug was described in terms of the biexponential expression: Cp = Ae-alphat + Be-betat. Based on total (free and bound) sulfonamide levels in the plasma, pseudodistribution equilibrium was slowly attained and the half-time of elimination (half-life) was 10.16 h +/- 2.50 (S.D., n = 6). Body clearance, which is the sum of all clearance processes, was 18.8 +/- 4.6 ml kg-1 h-1. Plasma protein binding, measured by equilibrium dialysis at sulfonamide concentration of 50 microgram/ml, was extensive (87.5% +/- 6.3, n =10). Computer-generated curves for an animal representative of the group, based on individual rate constants associated with the two-compartment open model, showed that 12% and 5% of the dose were present in the central and peripheral compartments, respectively, 24 h after administering the drug. A satisfactory dosage regimen might consist of a priming dose (55 mg/kg) and maintenance dosage (27.5 mg/kg at 24 h dosage intervals). Predicted plasma sulfadimethoxine concentrations would oscillate between 125 and 25 microgram/ml during the steady state. Influence of bacterial disease and febrile states on predicted levels remains to be verified experimentally.     

Betaine analogues alter homocysteine metabolism in rats

Glycine betaine supplementation lowers homocysteine levels in homocystinuria and in chronic renal failure patients through methylation catalysed by betaine-homocysteine methyltransferase (BHMT). The aim of this study was to determine the effect of glycine betaine analogues on homocysteine metabolism in Lewis rats. Glycine betaine, proline betaine, trigonelline, dimethylsulfoniopropionate (DMSP) or dimethylthetin (1.5 mmoles) was subcutaneously administered to rats fed a low betaine diet. The effect of each betaine on total plasma homocysteine and urinary and plasma betaine concentrations was monitored for 24h following administration. Baseline plasma homocysteine was 8.5 +/- micromol/l (S.E.M., n=44) and compared to controls concentrations decreased following glycine betaine (0.8+/-0.4 micromol/l, P = 0.064), DMSP (1.0+/-0.5 micromol/l, P = 0.041) and dimethylthetin (1.5 +/- 0.7micromol/l, P = 0.033) treatment, while concentrations increased following proline betaine (2.24 +/-0.7micromol/l, P = 0.002) and trigonelline (1.6 +/-0.3 micromol/l, P < 0.001) treatment. The effect of glycine betaine, DMSP and dimethylthetin on circulating homocysteine concentrations was thought to be mediated by BHMT in vivo. This hypothesis was supported by the finding that circulating glycine betaine concentrations increased following DMSP and dimethylthetin treatment. Proline betaine and trigonelline appeared to be poor BHMT substrates, being largely excreted in the urine unchanged, yet increased circulating homocysteine levels. This suggests they are inhibitors of BHMT. Urinary excretion of glycine betaine increased following treatment with all betaines, suggesting that the resorption of glycine betaine in the kidney was inhibited. The study shows that glycine betaine analogues have multiple effects on homocysteine metabolism (250).     

Radiation inactivation of multispecific transport systems for bile acids and xenobiotics in basolateral rat liver plasma membrane vesicles.

The functional molecular mass of the cholate, phallotoxin, iodipamide, and ouabain transport proteins in isolated basolateral plasma membrane vesicles was determined by radiation inactivation. Purified basolateral plasma membrane vesicles were irradiated (-90 to -120 degrees C) with high energy electrons from a 10-MeV linear accelerator at doses from 0 to 30 megarads. After each dose, the initial uptake, the equilibrium binding, and the binding of the substrates at 4 degrees C were checked. The size of the transporting function was, for cholate, 107 +/- 8.9 kDa; for phallotoxin, 104 +/- 7 kDa; and for ouabain, 120 +/- 4.7 kDa. The target size for the binding proteins was 56 +/- 4.2, 57 +/- 5, and 47.2 +/- 1.95 kDa for cholate, phallotoxin, and taurocholate, respectively. In the case of iodipamide, the functional molecular mass for both the transport and binding proteins was 54 +/- 4.8 kDa.     

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.     

Equilibrium binding of single-stranded DNA with herpes simplex virus type I-coded single-stranded DNA-binding protein, ICP8

We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I, in order to determine the thermodynamic parameters for its interaction with ssDNA. Fluorescence anisotropy measurements of a 5'-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25 degrees C, K = 0.55 +/- 0.05 x 10(6) M(-1), and a cooperativity parameter, omega = 15 +/- 3. The equilibrium constant was largely independent of salt, deltalog(Komega)/deltalog([NaCl]) = -2.4 +/- 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism.     

S-linked protein homocysteinylation: identifying targets based on structural, physicochemical and protein–protein interactions of homocysteinylated proteins

An elevated level of homocysteine, a thiol-containing amino acid is associated with a wide spectrum of disease conditions. A majority (>80 %) of the circulating homocysteine exist in protein-bound form. Homocysteine can bind to free cysteine residues in the protein or could cleave accessible cysteine disulfide bonds via thiol disulfide exchange reaction. Binding of homocysteine to proteins could potentially alter the structure and/or function of the protein. To date only 21 proteins have been experimentally shown to bind homocysteine. In this study we attempted to identify other proteins that could potentially bind to homocysteine based on the criteria that such proteins will have significant 3D structural homology with the proteins that have been experimentally validated and have solvent accessible cysteine residues either with high dihedral strain energy (for cysteine–cysteine disulfide bonds) or low pKa (for free cysteine residues). This analysis led us to the identification of 78 such proteins of which 68 proteins had 154 solvent accessible disulfide cysteine pairs with high dihedral strain energy and 10 proteins had free cysteine residues with low pKa that could potentially bind to homocysteine. Further, protein–protein interaction network was built to identify the interacting partners of these putative homocysteine binding proteins. We found that the 21 experimentally validated proteins had 174 interacting partners while the 78 proteins identified in our analysis had 445 first interacting partners. These proteins are mainly involved in biological activities such as complement and coagulation pathway, focal adhesion, ECM-receptor, ErbB signalling and cancer pathways, etc. paralleling the disease-specific attributes associated with hyperhomocysteinemia.