Acid glycohydrolase in Chinese hamster with spontaneous diabetes VII. The lack of short-term glucose-effect in cultured kidney cells

An epithelial cell line, designated CHK-AC_E, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-AC_E was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-AC_E-100 cultures grown in 100 and 400 mg/dl glucose for one passage.     

Peptide receptors in human lung tumor cells in culture: vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines

We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. ¹²⁵I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of ¹²⁵I-VIP in the 10^?10?10^?7M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.34}\text{nM}$) and 1062 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 61.4}\text{nM}$). Calu-1 cells have 36 300 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.33}\text{nM}$) and 1148 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 78.6}\text{nM}$). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.     

[3H] BOC [NLE28,31]CCK27−33, a new highly labelle ligand for CCK receptors: Binding on brain and on pancreas

Preliminary results on the binding of [³H]Boc[Nle^28,31]CCK_27^?33, designated [³H]Boc[diNle]CCK₇, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK₇ displaces [¹²⁵I]CCK₈ from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK₈. The order of potencies to displace [³H]Boc[diNle]CCK₇ from its binding sites was the same on mouse brain and rat pancreas: $\text{[}{}^3\text{HBoc[diNle]CCK}{}_7\text{>}\text{CCK}{}_8\text{,}\text{Boc-CCK}{}_7\text{>}\text{non-sulfated CCK}{}_8$, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [³H]Boc[diNle]CCK₇ is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments.     

Binding of the structural protein soc to the head shell of bacteriophage T4

Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N⁴-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C₃), -A-U-G-A- (mutant C₄) and -A-U-A-A- (mutant $\text{C}{}_3\text{C}{}_4$) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C₃), 3.2 (C₄) and 0.3 ($\text{C}{}_3\text{C}{}_4$). The finding that mutant C₃ no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C₄ RNA bound more efficiently than wild type, and that mutant $\text{C}{}_3\text{C}{}_4$ RNA showed substantial ribosome binding capacity whereas the single mutant C₃ did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of $\text{C}{}_3\text{C}{}_4$ this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.     

Phosphate transport in Neurospora: Kinetic characterization of a constitutive,low-affinity transport system

Log-phase cells of Neurospora crassa, grown in standard minimal medium, possess an energy-dependent transport system for inorganic phosphate, with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ (at pH 5.8) of 0.123 mM and a J_max of 1.64 mmoles/l cell water per min. Like the PO₄^3? transport system in yeast, the Neurospora system is stimulated by high intracellular K⁺. In addition, it is inhibited by high extracellular salt concentrations, an effect which may be related to the known depolarization of the Neurospora plasma membrane at high salt concentrations.The most striking property of the system is its strong dependence upon the extracellular pH. From pH 4.0 to pH 7.3, the J_max remains essentially constant but the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increases nearly 400-fold, from 0.01 to 3.62 mM. The increase cannot be accounted for by a single system with a preference for H₂PO₄^? (which would show only a 3-fold increase in apparent $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ over this pH range) nor by two systems with different affinities and pH optima (which would display nonlinear double-reciprocal plots at intermediate pH values). It can be explained, however, by a model in which OH^? or H⁺ is assumed to act as a modifier of the transport system, altering its affinity for substrate.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Oxidation of anionic nitroalkanes by flavoenzymes,and participation of superoxide anion in the catalysis

The reactivities of anionic nitroalkanes with 2-nitropropane dioxygenase of Hansenula mrakii, glucose oxidase of Aspergillus niger, and mammalian d-amino acid oxidase have been compared kinetically. 2-Nitropropane dioxygenase is 1200 and 4800 times more active with anionic 2-nitropropane than d-amino acid oxidase and glucose oxidase, respectively. The apparent K_m values for anionic 2-nitropropane are as follows: 2-nitropropane dioxygenase, 1.61 mm; glucose oxidase, 16.7 mm; and d-amino acid oxidase, 11.1 mm. Anionic 2-nitropropane undergoes an oxygenase reaction with 2-nitropropane dioxygenase and glucose oxidase, and an oxidase reaction with d-amino acid oxidase. In contrast, anionic nitroethane is oxidized through an oxygenase reaction by 2-nitropropane dioxygenase, and through an oxidase reaction by glucose oxidase. All nitroalkane oxidations by these three flavoenzymes are inhibited by Cu and Zn-superoxide dismutase of bovine blood, Mn-superoxide dismutases of bacilli, Fe-superoxide dismutase of Serratia marcescens, and other $\text{O}{}_2{}^{\mathrm{?}}$ scavengers such as cytochrome c and NADH, but are not affected by hydroxyl radical scavengers such as mannitol. None of the $\text{O}{}_2{}^{\mathrm{?}}$ scavengers tested affected the inherent substrate oxidation by glucose oxidase and d-amino acid oxidase. Furthermore, the generation of $\text{O}{}_2{}^{\mathrm{?}}$ in the oxidation of anionic 2-nitropropane by 2-nitropropane dioxygenase was revealed by ESR spectroscoy. The ESR spectrum of anionic 2-nitropropane plus 2-nitropropane dioxygenase shows signals at g₁ = 2.007 and g₁₁ = 2.051, which are characteristic of $\text{O}{}_2{}^{\mathrm{?}}$. The $\text{O}{}_2{}^{\mathrm{?}}$ generated is a catalytically essential intermediate in the oxidation of anionic nitroalkanes by the enzymes.     

Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

The turnover of acetylcholine receptors labeled with ¹²⁵I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≥ 5}\text{days}$) and different than the rate of extrajunctional receptor degradation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

Inactivation of the human CuZn superoxide dismutase during exposure to O-2 and H2O2

Human copper-zinc superoxide dismutase undergoes inactivation when exposed to O₂^? and H₂O₂ generated during the oxidation of acetaldehyde by xanthine oxidase at pH 7.4 and 37° C. In contrast, human manganese superoxide dismutase is not inactivated under the same conditions. Catalase and Mn-superoxide dismutase protect CuZn superoxide dismutase from inactivation. Similar protection is observed with hydroxyl radical (OH.) scavengers, such as formate and mannitol. In contrast, other OH. scavengers such as ethanol and tert-butyl alcohol, have no protective action. The latter results indicate that “free OH.” is not responsible for the inactivation. Furthermore, H₂O₂ generated during the oxidation of glucose by glucose oxidase, i.e., without production of O₂^?, does not induce CuZn superoxide dismutase inactivation. A mechanism accounting for this $\text{O}{}_2{}^{\mathrm{?}}\text{H}{}_2\text{O}{}_2$-dependent inactivation of CuZn superoxide dismutase is proposed.     

Developmental biochemistry of cotton seed embryogenesis and germination. VII. Characterization of the cotton genome.

The DNA of cotton, Gossypium hirsutum, has been characterized as to spectral characteristics, buoyant density in CsCl, base composition, and genetic complexity. The haploid genome size is found to bo 0.795 pg DNA/cell. However, the amount of DNA per cell in the cotyledons increases during embryogenesis to an average ploidy level of 12N in the mature seed cotyledons. Reassociation kinetics indicate that this increase is due to endoreduplication of the entire genome.Non-repetitive deoxynucleotide sequences account for approximately 60.5% of the cotton genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$pure⁵ = 437); highly repetitive sequences (> 10,000 repetition frequency) constitute about 7.7% of the genome. ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 4.6 × 10}{}^{\mathrm{?4}}$) and intermediately repetitive sequences constitute the remaining 27% of the genome ($\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{pure}\text{= 1.46}$). Hybridization of ¹²⁵I-labeled cytoplasmic ribosomal RNA to whole-cell DNA on filters and in solution indicate approximately 300 to 350 copies of the rRNA cistrons per haploid genome.The interspersion of repetitive sequences that reassociate between C₀t values of 0.1 and 50 with non-repetitive sequences of the cotton genome has been examined by determining the reassociation kinetics of DNA of varying fragment lengths and by the electron microscopy of reassociated molecules. About 60% of the genome consists of non-repetitive regions that average 1800 base-pairs interpersed with repetitive sequences that average 1250 base-pairs. Approximately 20% of the genome may be involved in a longer period interspersion pattern containing non-repetitive sequences of approximately 4000 base-pairs between repetitive sequences. Most of the individual sequences of the interspersed repetitive component are much smaller than the mass average size, containing between 200 and 800 base-pairs. Sequence divergence is evident among the members of this component.Highly repetitive sequence elements that are reassociated by a C₀t value of 0.1 average 2500 base-pairs in length, appear to have highly divergent regions and do not appear to be highly clustered. A portion of this highly repetitive component reassociates by C₀t = 10^?4, zero-time binding DNA, and accounts for less than 3% of the genome. At least a third of these sequences appear by electron microscopy to be intramolecular duplexes (palindromes) of 150 to 200 base-pairs and to occur in clusters.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

beta-Endorphin: characteristics of binding sites in the rat brain.

Stereospecific binding of human β-endorphin to rat membrane preparations is described for the first time using $\text{[}{}^3\text{H-Tyr}{}^{27}\text{]-β}{}_{\mathrm{h}}\text{-endorphin}$ as the ligand. The binding is time dependent and saturable with respect to β_h-endorphin with an apparent dissociation constant of 0.3 nM. Sodium ion (100 mM) elevates this value to 2.5 nM but has no effect on the total number of binding sites present in the membrane preparation. The ability of certain β-endorphin analogs, opiate agonists as well as antagonists to inhibit the binding of β_h-endorphin, is presented.     

Association of α2-macroglobulin-thrombin and α2-macroglobulin-plasmin complexes with isolated hepatocytes

¹²⁵I-labelled $\text{α}{}_2\text{-}\text{macroglobulin}$ complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values calculated from displacement experiments were 7.9 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ and 8.5 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. $\text{α}{}_2\text{-}\text{Macroglobulin}$ itself bound also to hepatocytes, but the affinity of the $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes was higher than that of the inhibitor alone, and $\text{α}{}_2\text{-}\text{macroglobulin}$ was not internalized, either. ¹²⁵I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes. The $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ complex competed with the $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$ complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for $\text{α}{}_2\text{-}\text{macroglobulin-proteinase}$ and antithrombin III-proteinase complexes are different.     

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of ¹²⁵I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, $\text{K}\text{?}{}_{\mathrm{<mtext>e</mtext>}}$. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for K_d. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.