Pure Noncompetitive Inhibition Research Articles

ERRATUM: Evaluation of the Anti-inflammatory, Antioxidant, and Protease Inhibitory Activity of the Crude Methanol Extract of Portulaca oleracea

Protease inhibitors have considerable applications in biomedicine, biotechnology, and therapy. The aim of the study was to evaluate the methanol crude extracts of aerial portions of Portulaca oleracea (P. oleracea) for anti-inflammatory, antioxidant, and protease inhibitor activities, and to evaluate their inhibitory action against various therapeutically significant proteases. A modified colorimetric assay was used to analyze the protease inhibitors on trypsin and other therapeutic enzymes. P. oleracea crude leaf extract exhibited 79.92% antioxidant and 75% anti-inflammatory activity. Analysis of the effects of crude P. oleracea extract on the values of enzyme kinetics (Km, Vmax) was determined by Lineweaver-Burk reciprocal plot. It was found that the extract exhibited a potent inhibitory effect on the Vmax (µmole/min) of the chymotrypsin 26.0, trypsin 18.0, papain 8.33, and elastase 10.0, when compared to control 41.0, 73.0, 11.11, and 20.3, respectively. While no effect on Km except for elastase which was decreased from 0.026 mM for the control to 0.01 mM. It was found that this effect showed a powerful inhibition in a different way. There is a mixed non-competitive inhibition on elastase as well as pure non-competitive inhibition on chymotrypsin, trypsin, and papain. The crude extract contained 53.3%, 55.7%, 14.9%, and 26.9%, respectively, of protease inhibitors for the therapeutic protease enzymes trypsin, papain, chymotrypsin, and elastase. Therefore, trypsin, papain, chymotrypsin, and elastase have been inhibited by the crude extract of P. oleracea. Characterization of the crude extract indicated potent anti-inflammatory and antioxidant effects.

Multimodal α-Glucosidase and α-Amylase Inhibition and Antioxidant Effect of the Aqueous and Methanol Extracts from the Trunk Bark of Ceiba pentandra.

Postprandial hyperglycemia and oxidative stress are important factors that worsen the health condition of patients with type 2 diabetes. We recently showed that extracts from Ceiba pentandra mitigate hyperglycemia in dexamethasone- and high diet/streptozotocin-induced diabetes. Herein, we evaluated the postprandial regulatory properties and the antioxidant effects of the aqueous (AE) and methanol (ME) extracts from the stem bark of Ceiba pentandra. The phytochemical analysis of AE and ME was performed using the LC-MS technique and the total phenolic and flavonoid assays. Both extracts were tested for their ability to inhibit superoxide anion (O2•ـ), hydrogen peroxide (H2O2), protein oxidation, alpha-amylase, and alpha-glucosidase activities. The mode of enzyme inhibition was also determined in a kinetic study. AE and ME were both rich in phenolic and flavonoid compounds. ME was 2.13 and 1.91 times more concentrated than AE in phenolic and flavonoid compounds, respectively. LC-MS allowed the identification of 5 compounds in both extracts. ME and AE inhibited O2•ـ with IC50 of 51.81 and 34.26 μg/ml, respectively. On H2O2, they exhibited IC50 of 44.84 and 1.78 μg/ml, respectively. Finally, they exhibited IC50 of 120.60 and 140.40 μg/ml, respectively, in the inhibition of protein oxidation induced by H2O2, while showing IC50 of 39.26 and 97.95 μg/ml on the protein oxidation induced by AAPH. ME and AE inhibited alpha-amylase with IC50 of 6.15 and 54.52 μg/ml, respectively. These extracts also inhibited alpha-glucosidase, demonstrating IC50 of 76.61 and 86.49 μg/ml. AE exhibited a mixed noncompetitive inhibition on both enzymes, whereas ME exhibited a competitive inhibition on α-amylase and a pure noncompetitive inhibition on α-glucosidase. These results demonstrate that ME and AE scavenge reactive oxygen species and prevent their effects on biomolecules. Besides, ME and AE inhibit carbohydrate digestive enzymes. These properties may contribute to reduce postprandial hyperglycemia and regulate glycemia in diabetic patients.

Characterization of WY 14,643 and its Complex with Aldose Reductase.

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.

Property of midgut α-amylase from Mythimna separata (Lepidoptera: Noctuidae) larvae and its responses to potential inhibitors in vitro.

Midgut α-amylase is an important digestive enzyme involved in larval energy metabolism and carbohydrate assimilation. In this article, the properties of midgut α-amylase from the Oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae), larvae were characterized, and its in vitro responses to chemical inhibitors were also determined. The kinetic parameters Km and Vmax of midgut α-amylase were 0.064 M , 4.81 U mg pro −1 in phosphate buffer, and 0.128 M , 1.96 U mg pro −1 in barbiturate-acetate buffer; α-amylase activity linearly increased as starch concentration increased. α-Amylase activity was not influenced by amino acids such as Pro, Met, Try, His, Ala, and Phe but was strongly activated by antioxidants. Reduced glutathione, 1,4-dithiothreitol, β-mercaptoethanol, and ascorbic acid improved the activity of α-amylase about 2.06, 3.46, 3.37, and 6.38 times, respectively, relative to the control. Ethylenediaminetetraacetic acid, sodium dodecyl sulfonate, and N -bromosuccinimide (NBS) strongly inhibited α-amylase. α-, β-, and γ-cyclodextrin were not the preferred substrates for α-amylase. Kinetic analysis showed that IC 50 value of NBS against α-amylase was 1.52 (±0.26) µM, and the mode of action of NBS with Ki as 2.53 (0.35) µM was a mixed-type inhibition that indicated a combination of partial competitive and pure noncompetitive inhibition. The midgut α-amylase of armyworm larvae may be a potential target for novel insecticide development and pest control.

Kinetic Mechanism of Protein Arginine Methyltransferase 6 (PRMT6)

The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyze the mono- and dimethylation of arginine residues in a variety of proteins. Although these enzymes play important roles in a variety of cellular processes, aberrant PRMT activity is associated with several disease states, including heart disease and cancer. In an effort to guide the development of inhibitors targeting individual PRMTs, we initiated studies to characterize the molecular mechanisms of PRMT catalysis. Herein, we report studies on the kinetic mechanism of PRMT6. Initial velocity, product inhibition, and dead-end analog inhibition studies with the AcH4-21 and R1 peptides, as well as their monomethylated versions, indicate, in contrast to a previous report, that PRMT6 utilizes a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes.

Mechanism of cytochrome P450-3A inhibition by ketoconazole

Ketoconazole is extensively used as an index inhibitor of cytochrome P450-3A (CYP3A) activity in vitro and in vivo, but the mechanism of ketoconazole inhibition of CYP3A still is not clearly established. Inhibition of metabolite formation by ketoconazole (seven concentrations from 0.01 to 1.0 µm) was studied in human liver microsomes (n = 4) at six to seven substrate concentrations for triazolam, midazolam, and testosterone, and at two substrate concentrations for nifedipine. Analysis of multiple data points per liver sample based on a mixed competitive-noncompetitive model yielded mean inhibition constant K(i) values in the range of 0.011 to 0.045 µm. Ketoconazole IC50 increased at higher substrate concentrations, thereby excluding pure noncompetitive inhibition. For triazolam, testosterone, and midazolam α-hydroxylation, mean values of α (indicating the 'mix' of competitive and noncompetitive inhibition) ranged from 2.1 to 6.3. However, inhibition of midazolam 4-hydroxylation was consistent with a competitive process. Determination of K(i) and α based on the relation between 50% inhibitory concentration values and substrate concentration yielded similar values. Pre-incubation of ketoconazole with microsomes before addition of substrate did not enhance inhibition, whereas inhibition by troleandomycin was significantly enhanced by pre-incubation. Ketoconazole inhibition of triazolam α- and 4-hydroxylation, midazolam α-hydroxylation, testosterone 6β-hydroxylation, and nifedipine oxidation appeared to be a mixed competitive-noncompetitive process, with the noncompetitive component being dominant but not exclusive. Quantitative estimates of K(i) were in the low nanomolar range for all four substrates.

Control and signal processing by transcriptional interference

A transcriptional activator can suppress gene expression by interfering with transcription initiated by another activator. Transcriptional interference has been increasingly recognized as a regulatory mechanism of gene expression. The signals received by the two antagonistically acting activators are combined by the polymerase trafficking along the DNA. We have designed a dual-control genetic system in yeast to explore this antagonism systematically. Antagonism by an upstream activator bears the hallmarks of competitive inhibition, whereas a downstream activator inhibits gene expression non-competitively. When gene expression is induced weakly, the antagonistic activator can have a positive effect and can even trigger paradoxical activation. Equilibrium and non-equilibrium models of transcription shed light on the mechanism by which interference converts signals, and reveals that self-antagonism of activators imitates the behavior of feed-forward loops. Indeed, a synthetic circuit generates a bell-shaped response, so that the induction of expression is limited to a narrow range of the input signal. The identification of conserved regulatory principles of interference will help to predict the transcriptional response of genes in their genomic context.

Regulation of Escherichia coli Polynucleotide Phosphorylase by ATP

Polynucleotide phosphorylase (PNPase), an enzyme conserved in bacteria and eukaryotic organelles, processively catalyzes the phosphorolysis of RNA, releasing nucleotide diphosphates, and the reverse polymerization reaction. In Escherichia coli, both reactions are implicated in RNA decay, as addition of either poly(A) or heteropolymeric tails targets RNA to degradation. PNPase may also be associated with the RNA degradosome, a heteromultimeric protein machine that can degrade highly structured RNA. Here, we report that ATP binds to PNPase and allosterically inhibits both its phosphorolytic and polymerization activities. Our data suggest that PNPase-dependent RNA tailing and degradation occur mainly at low ATP concentrations, whereas other enzymes may play a more significant role at high energy charge. These findings connect RNA turnover with the energy charge of the cell and highlight unforeseen metabolic roles of PNPase.

Mechanistic characterization for c-jun-N-Terminal Kinase 1α1

c-jun-N-terminal kinase 1α1 (JNK1α1) is a serine/threonine kinase of the mitogen-activated protein (MAP) kinase family that phosphorylates protein transcription factors after activation by a variety of environmental stressors. In this study, the kinetic mechanism for JNK1α1 phosphorylation of activating transcription factor 2 (ATF2) was determined utilizing steady-state kinetics in the presence and absence of both ATF2 and ATP competitive inhibitors. Data from initial velocity studies were consistent with a sequential mechanism for JNK1α1. AMP-PCP exhibited competitive inhibition versus ATP and pure noncompetitive inhibition versus ATF2. JIP-1 peptide (RPKRPTTLNLF) was competitive versus ATF2 and mixed noncompetitive versus ATP. These data suggest that JNK1α1 proceeded via a random sequential kinetic mechanism with non-interacting ATF2 and ATP substrate sites.

Kinetic Mechanism and Inhibitor Characterization for c-jun-N-Terminal Kinase 3α1

c-jun-N-Terminal kinase 3alpha1 (JNK3alpha1) is a mitogen-activated protein (MAP) kinase family member expressed primarily in the brain that phosphorylates protein transcription factors including c-jun and activating transcription factor 2 (ATF2) upon activation by a variety of stress-based stimuli. In this study, the kinetic mechanism for JNK3alpha1 was determined via initial velocity patterns in the presence and absence of both ATP and ATF2 competitive inhibitors. Peptide inhibitors from both ATF2 (peptide 1) and JNK-interacting protein 1 (JIP-1) (peptide 3), derived from the homologous delta-domain JNK docking sequence, inhibited JNK3alpha1 activity in a competitive fashion versus ATF2 while being pure noncompetitive toward ATP. In contrast, peptides derived from the phosphoacceptor activation domain on ATF2 (peptides 4 and 5) were recognized neither as substrates nor as inhibitors of JNK3alpha1. AMP-PCP and compound 6, a small molecule analinopyrimidine, exhibited pure noncompetitive inhibition versus ATF2 and competitive inhibition versus ATP. Peptide inhibitors based on the delta-domain sites of JIP ( 3) and ATF2 ( 1) were not recognized by p38, also of the MAPK family, which may give insight into finding more selective inhibitors for the JNK family of kinases. Collectively these data showed that JNK3alpha1 proceeded by a random sequential kinetic mechanism and that the ATP and ATF2 substrate sites were non-interacting. Moreover, these results established the 11-mer JIP peptide ( 3) as a potent ( K i = 25 +/- 6 nM) competitive inhibitor versus ATF2 in JNK3alpha1.

Inhibiting wild-type and C299S mutant AKR1B10; a homologue of aldose reductase upregulated in cancers

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

Use of Substrate Analogs and Mutagenesis to Study Substrate Binding and Catalysis in the Sir2 Family of NAD-dependent Protein Deacetylases

The Sir2 family of enzymes is highly conserved throughout evolution and functions in silencing, control of life span, apoptosis, and many other cellular processes. Since the discovery of the NAD-dependent deacetylase activity of Sir2 proteins, there has been a flurry of activity aiming to uncover the mode of substrate binding and catalysis. Structural and biochemical studies have led to several proposed reaction mechanisms, yet the exact catalytic steps remain unclear. Here we present in vitro studies of yeast homolog Hst2 that shed light on the mechanism of Sir2 proteins. Using acetyl-lysine substrate analogs, we demonstrate that the Hst2 reaction proceeds via an initial SN2-type mechanism with the direct formation of an ADP-ribose-acetyl-lysine intermediate. Kinetic studies further suggest that ADP-ribose inhibits the Hst2 reaction in a biologically relevant manner. Through biochemical and kinetic analyses of point mutants, we also clarify the role of several conserved core domain residues in substrate binding, stabilization of the ADP-ribose-acetyl-lysine intermediate, and catalysis. These findings bring us a few steps closer to understanding Sir2 activity and may provide a useful platform for the design of Sir2-specific inhibitors for analysis of Sir2 function and possibly therapeutic applications.

WY 14,643 inhibits human aldose reductase activity

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. Our data reveal that peroxisome proliferator WY 14,643, follows a pure non-competitive inhibition pattern in the aldehyde reduction activity as well as in the alcohol oxidation activity of AR. This finding communicates for the first time a novel feature of WY 14,643 in regulating AR activity. In addition, this observation indicates that AR, AR-like proteins and aldo-keto reductase (AKR) members may be involved in the WY 14,643 mechanism of action when it is administered as PPAR agonist.

Fibrates inhibit aldose reductase activity in the forward and reverse reactions

Fibrates such as bezafibrate, gemfibrozil, clofibric acid, ciprofibrate and fenofibrate, are ligands for peroxisome proliferator-activated receptor α (PPARα), and are used as therapeutic agents in the treatment of hyperlipidemia. Synthesis and accumulation of sorbitol in cells due to aldose reductase (AR) activity is implicated in secondary diabetic complications. In pursuit of finding a lead compound identification to design an effective AR inhibitor employing fragment-based design-like approach, we found that this class of compounds and their nearest neighbors could inhibit AR. Bezafibrate and gemfibrozil displayed a mixed non-competitive inhibition pattern in the glyceraldehyde reduction activity and pure non-competitive inhibition pattern in the benzyl alcohol oxidation activity of AR. Clofibric acid, ciprofibrate and fenofibrate showed pure non-competitive inhibition patterns in the forward reaction. In the reverse reaction, clofibric acid displayed a non-competitive inhibition pattern while ciprofibrate and fenofibrate displayed competitive inhibition patterns. This finding reveals for the first time a novel attribute of the fibrates in the regulation of AR activity and may be useful as lead compounds to control the function of AR in the progression and treatment of secondary diabetic complications in addition to other clinical conditions. Alternatively, these findings demonstrate that AR plays a significant role in the fibrate metabolism under various scenarios.

Regulation of AMP Deaminase by Phosphoinositides

AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K(i) of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)X(n)(R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.

Oxime Inhibition of Nitrification During Treatment of an Ammonia‐Containing Industrial Wastewater

Studies were conducted to investigate the nitrification potential of an industrial wastewater that contained high amounts of ammonium sulfate. Laboratory‐scale, activated‐sludge reactors were unable to nitrify the wastewater, but the same sludge began to nitrify within 2 weeks after the feed was changed to a synthetic wastewater. Further investigation indicated that oxime compounds in the industry's wastewater were causing nitrification inhibition. Four oximes known to exist in the industrial wastewater were studied: cyclohexanone oxime was not inhibitory up to 100 mg/L, acetaldehyde oxime and aldecarb oxime were moderately inhibitory, and methylethyl ketoxime (MEKO) was strongly inhibitory to nitrification. The mechanism of MEKO inhibition was evaluated using three different analytical approaches. Methylethyl ketoxime demonstrated pure noncompetitive inhibition patterns using all methods of data analysis. The inhibition constant Ki indicates the concentration that causes 50% inhibition and was estimated to range from 0.3 to 1.9 mg/L at 25 °C for MEKO.

On the inhibition of camel retina acetylcholinesterase activity by cycloheximide in vitro.

The kinetic parameters of inhibition of camel retinal acetylcholinesterase (AChE) activity by cycloheximide (CH) were investigated. For the control system, the Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.076 mmol/L and the Vmax was 0.547 mumol/min per mg protein. In contrast, these parameters were decreased in the CH-treated systems. Dixon and Lineweaver-Burk plots, and their secondary replots, indicated that the inhibition was of the linear mixed type, which seems to be a combination of partial competitive and pure noncompetitive inhibition. The values of K'i(slope) and KI(intercept) were estimated to be 3.50 and 5.68 mmol/L, respectively. Ki was greater than K'i, indicating that CH has a greater binding affinity for the peripheral site than the active site.

Effect of KW-3902, a novel adenosine A 1 receptor antagonist, on sodium-dependent phosphate and glucose transport by the rat renal proximal tubular

KW-3902, 1, 3-dipropyl-8-(3-noradamantanyl) xanthine, is a novel potent and selective adenosine A 1-receptor antagonist. KW-3902 has been found to cause significant diuresis and natriuresis. To investigate the action of this adenosine A 1-receptor antagonist on phosphate transport in renal proximal tubular cells, we studied its effect on the uptake of phosphate by the cultured rat renal proximal tubular cell. KW-3902 significantly inhibited sodium-dependent uptake of phosphate at 10 minutes. The inhibitory effect was dose-dependent with maximum effect achieved at a KW-3902 concentration of 3×10 −5 M in the uptake media. The half-maximal inhibitory concentration, IC 50, of KW-3902 on phosphate uptake was 2×10 −6 M. Dixon plot analysis op the uptake data was consistent with pure non-competitive inhibition. The inhibition constant, K i, of 6.2×10 −6 M for phosphate transport, derived from Dixon plot, was in close agreement with the IC 50 calculated from a semilog dose response curve. Sodium-dependent glucose transport was not affected by KW-3902. These findings reveal that KW-3902 has a direct and specific inhibitory effect on phosphate uptake in renal proximal tubules.

Inhibitors of human immunodeficiency virus-1 protease

We have shown that the interaction of pepstatin A with human immunodeficiency virus-1 protease (HIV-1 protease) can be characterized by a high-affinity mode (Ki = 478 +/- 27 nM), resulting in pure competitive inhibition of the hydrolytic activity of HIV-1 protease toward the fluorogenic substrate. Binding of pepstatin in this mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 10 microM pepstatin. Haloperidol, in contrast, interacts with HIV-1 protease with weaker affinity (Ki = 19 +/- 1 microM) in a mode which results in pure noncompetitive inhibition of the hydrolytic activity of HIV-1 protease. Binding of haloperidol in this mode induces a red shift in the endogenous fluorescence arising from the tryptophan residues in HIV-1 protease. This shift is maximal in the presence of 200 microM haloperidol. Addition of both pepstatin and haloperidol at concentrations in the range of their Ki values results in additive inhibition of the hydrolytic activity of HIV-1 protease, as well as an additive effect on the tryptophan fluorescence of protease. However, at saturating concentrations of pepstatin and haloperidol, the effect of haloperidol was predominant, as measured by the changes in the intrinsic fluorescence of HIV-1 protease.

Effect of alpha interferon on glucose and alanine transport by rat renal brush border membrane vesicles

To investigate the pathogenetic mechanisms of interferon nephrotoxicity, we studied the effect of recombinant interferon alfa-2b on the uptake of 14C-D-glucose and 14C-L-alanine by rat renal brush-border-membrane vesicles. Interferon significantly inhibited 20 sec. sodium-dependent and 5 and 10 min. equilibrium uptake of both glucose and alanine. The inhibitory effect was dose dependent with maximum effect achieved at interferon concentration of 5 × 10 −8M in the uptake media. The half-maximal inhibitory concentrations, IC 50, of interferon on glucose uptake was 1.8 × 10 −8M, and 5.4 × 10 −9M on alanine uptake. Dixon plot analysis of uptake data was consistent with pure non-competitive inhibition. The inhibition constants, K i, 1.5 × 10 −8M for glucose uptake, and 7.3 × 10 −9M for alanine uptake, derived from Dixon plots were in close agreement with the IC 50s calculated from the semilog dose response curves. These observations reveal that direct interactions at the proximal tubule cell membrane are involved in the pathogenesis of interferon nephrotoxicity, and that its mechanism of nephrotoxicity is similar to that of other low molecular weight proteins.