Cytoplasmic aldo-keto reductases: a class of drug metabolizing enzymes.

Aldehyde and ketone xenobiotic substances are preferentially reduced to alcohols by cytoplasmic enzymes in mammals. These enzymes are widely distributed in the tissues, have broad substrate specificities, have similar physical-chemical characteristics, and require reduced nicotinamide adenine dinucleotide as cofactor for the reductions. These reductases define a system of detoxification for aldehyde and ketone groups.

Diurnal rhythm: effects on hepatic regeneration and hepatic regenerative stimulator substance.

The effect of a controlled lighting schedule on the activity of a weanling rat liver extract that stimulates DNA synthesis in regenerating adult rat liver, and on the response of the test animals to the extract, has been investigated. Both activity of the extract and endogenous DNA synthesis in the weanling animals follow the same distinct diurnal rhythm. Reversal of the lighting schedule reverses the rhythm of endogenous DNA synthesis but activity of the extract no longer correlates with the peak of DNA synthesis. Diurnal rhythm also has a striking effect on DNA synthesis in the regenerating test animal, but the extract increases DNA synthesis to the same relative degree, regardless of the time of day the hepatectomy is performed.

Experimental chemotherapy-induced skin necrosis in swine. Mechanistic studies of anthracycline antibiotic toxicity and protection with a radical dimer compound.

The reactivity of antitumor anthracycline and mitomycin C antibiotics with the oxomorpholinyl radical dimers, bi(3,5,5-trimethyl-2-oxomorpholin-3-yl) (TM3) and bi(3,5-dimethyl-5-hydroxymethyl-2-oxomorpholin-3-yl) (DHM3), was studied in vitro. The oxomorpholinyl radical reduced daunorubicin to a quinone methide intermediate that reacted with solvent to form 7-deoxydaunorubicinone. The solvolysis reaction followed first order kinetics, and the reactivity rate constants (k2) measured for seven anthracycline analogues ranged from 2 X 10(-2) s-1 to 8.0 X 10(-4) s-1. The chemical reactivity of each anthracycline quinone methide correlated with the total skin toxicity caused by the respective parent anthracycline following injection into swine skin. Microscopic examination of experimental lesions in swine skin resemble those observed in humans after inadvertant chemotherapy extravasation. Hydrocortisone sodium succinate was not effective for the treatment of doxorubicin-induced skin necrosis, whereas DHM3 was effective for the treatment of skin necrosis caused by all seven anthracyclines and by the quinone containing antibiotic, mitomycin C.

Alterations in adriamycin efficacy by phenobarbital.

Adriamycin dosage should be reduced in patients with impaired liver function, since adriamycin disposition is influenced by liver metabolism and biliary excretion. It follows that drugs that increase the metabolism or excretory capacity of the liver may decrease adriamycin concentrations to suboptimal values. Adriamycin metabolism was therefore studied in mice pretreated with phenobarbital (75 mg/kg i.v.) by injection. After an i.v. dose of adriamycin (30 mg/kg i.v.), plasma fluorescence due to drug and metabolites was less and disappeared at a greater rate in phenobarbital-pretreated mice than control animals. When extracted with chloroform: isoprophyl alcohol (1:1), the livers from the phenobarbital-pretreated group yielded a greater concentration of glycones. Experiments with liver microsomes confirmed that aglycone production occurred at a more rapid initial rate in phenobarbital-induced livers. No increase in aldoketo reductase (daunorubicin reductase) activity was noted. Phenobarbital-pretreated mice, inoculated i.p. with 1 million L1210 cells and then treated with adriamycin (6 mg/kg i.v.), had significantly lower survival than did controls (p less than 0.01). These findings show that phenobarbital affects the disposition of adriamycin by microsomal enzyme induction and suggest that drugs that induce microsomal enzymes should not be used concurrently with adriamycin if optimal drug efficacy is desired.

Plasma pharmacokinetics of adriamycin and its metabolites in humans with normal hepatic and renal function.

A new, nondestructive, plasma extraction technique ultilizing chloroform:isopropyl alcohol (1:1) and ammonium sulfate saturation has been devised to isolate adriamycin and its metabolites from human plasma. Adriamycin was the most prominent species in plasma. It disappeared according to a triphasic pattern with a mean half-life of 30 hr. Six metabolites have been clearly separated from adriamycin by thin-layer chromatography. Three were aglycones and three were polar metabolites, one of which has been identified as adriamycinol. All metabolites appeared rapidly in plasma and disappeared according to a biphasic or tri-phasic pattern. The polar metabolites in plasma were found in similar relative concentration to those in urine. In contrast to the small Quantities of aglycones in urine, however, significant concentrations of aglycones were found in plasma. The least prominent metabolite was adriamycin aglycone; the most prominent metabolite was a less polar aglycone, most likely deoxyadriamycin aglycone, and a more polar aglycone, presumably demethyl deoxyadriamycinol aglycone, was the only metabolite to show variable pharmacokinetics in different patients. The nondestructive plasma extraction technique has verified the presence of extensive human metabolism of adriamycin and demonstrated the presence of aglycone and polar metabolites.

Nuclear catalyzed antibiotic free radical formation.

Nuclei isolated from rat liver, heart, and kidney catalyze oxygen consumption in the presence of reduced pyridine nucleotide (NADPH) and quinone or quinone-imine antibiotics such as Adriamycin, daunorubicin, actinomycin D, mitomycin C, and streptonigrin. The Km and Vmax values for NADPH were 2.4 x 10(-3) M and 3 x 10(-8) mol O2 per min per mg protein and Km values for the antibiotics ranged from 1.4 x 10(-4) M to 5.9 x 10(-6) M. Metabolism of the anthracycline antibiotics, i.e., reductive glycosidase reaction, occurs in reaction mixtures after all oxygen is consumed. During the reaction, free-radical species of Adriamycin and daunorubicin are detectable by electron paramagnetic reasonance spectrometry. These observations indicate that some cytotoxic antibiotics can be activated to a free-radical state at the site where damage to nuclear DNA may result.

Metabolism and disposition of menogaril (NSC 269148) in the rabbit.

We have investigated the metabolism and disposition, in rabbits, of menogaril (7-OMEN), a new anthracycline antibiotic recently introduced into clinical trials. 7-OMEN was administered by rapid i.v. injection at a dosage of 2.5 mg/kg. 7-OMEN and metabolites were assayed by high performance liquid chromatography. Plasma concentrations of 7-OMEN declined in biexponential fashion with a terminal half-life of 2.7 h. The area under the plasma concentration versus time curve was 1.3 microM X h. The systemic clearance of 7-OMEN was 57.6 ml/min/kg. No metabolite of 7-OMEN was detected in plasma. At 8 h after treatment, the cumulative urinary and biliary excretions of 7-OMEN equivalents amounted to 1.3 and 3.4% of the total administered dose, respectively. 7-OMEN was the predominant fluorescent compound in urine, but four metabolites were also seen. In bile, 7-OMEN represented only 9.6% of the cumulative excretion and six metabolites were observed. Among the organs, lungs contained the highest concentrations of parent drug. Substantial concentrations of metabolites were observed in the kidneys, liver, duodenum, and small intestine. Three of the observed metabolites of 7-OMEN have been tentatively identified as N-demethylmenogaril, 7-deoxynogarol, and N-demethyl-7-deoxynogarol.

Radical dimer rescue of toxicity and improved therapeutic index of adriamycin in tumor-bearing mice.

The product of adriamycin (ADR) reductive glycosidic cleavage is the pharmacologically inactive 7-deoxyadriamycin aglycone. Bi(3,5-dimethyl-5-hydroxymethyl-2-oxomorpholin-3-yl) (DHM3) is a radical dimer which reacts with ADR in vitro to produce this aglycone. We utilized DHM3 to prevent ADR toxicity in mice. CD2F1 male mice were given a single dose of ADR, 25 mg/kg i.p., which was acutely lethal as indicated by a median survival time of 7 days. DHM3 administered as a single i.p. dose of 50 mg/kg 15 or 30 min following ADR provided significant protection with median survival times greater than 9 wk. Mice bearing ascitic L1210 leukemic cells were given ADR, 0, 6.6, 15, or 25 mg/kg i.p. 1 day following inoculation of tumor. DHM3 administered as a single 50 mg/kg i.p. dose 20 min after ADR had no significant effect on ADR efficacy at the lower dose range (% treated versus control = 171 and 285 for 6.6 and 15.0 mg/kg, respectively). Less than 15% of the animals in these treatment groups were long-term survivors. However, following high doses of ADR (25 mg/kg), DHM3 protected mice from ADR lethality and over 70% of animals were long-term survivors. The determination of parent ADR and ADR aglycone content in several tissues indicated that the concentration of ADR was reduced in those animals that received DHM3 15 min after ADR. Correspondingly an increase in ADR aglycone concentration in each tissue resulted from DHM3 treatment. DHM3 represents a novel class of compounds that can ameliorate ADR toxicity and has potential use as a rescue agent.

Effects of deoxynucleosides on cultured human leukemia cell growth and deoxynucleotide pools.

We investigated the mechanism of cell growth inhibition caused by the deoxyribonucleosides thymidine (dThd), deoxyguanosine (dGuo), deoxyadenosine (dAdo), and deoxycytidine (dCyd). Growth of the cultured human leukemic cells HL-60 and K-562 was measured by cloning in soft agar. Of the deoxyribonucleosides, dGuo was the most potent cell growth inhibitor; however, the potency of added dAdo was probably attenuated by the presence of adenosine deaminase in the tissue culture growth medium. The concentrations of nucleoside causing 50% inhibition of HL-60 cloning were: dCyd, greater than 10,000 microM; dAdo, 500 microM; dThd, 5,000 microM; and dGuo, 80 microM. For K-562 cloning, the concentrations causing 50% inhibition of cloning were dCyd, greater 10,000 microM; dAdo, 1,600 microM; dThd, 880 microM;' and dGuo, 100 microM. Measurement of deoxycytidine 5'-triphosphate (dCTP) pool size in HL-60 cells following incubation with 750 microM deoxyribonucleosides revealed that dGuo caused the greatest reduction of dCTP pools, both in early (passage 10)- and late (passage 71)-passage-derived HL-60 cell cultures (35 and 19% of control, respectively), compared to dThd (61 and 26% of control, respectively) and dAdo (39% of control of HL-60 passage 10). In K-562 cells, reductions in dCTP pool size caused by dAdo, dThd, and dGuo were 68, 46, and 35% of control, respectively. Incorporation of [3H]dCyd into DNA of HL-60 and K-562 cells was enhanced by dThd and dGuo, but the degree of enhancement was greater for dThd than for dGuo. Despite its effect in reducing HL-60 dCTP pool size, dAdo failed to enhance [3H]dCyd incorporation in either HL-60 or K-562 cells. Addition of dCyd to the cultures could only partially rescue the inhibition of HL-60 cloning caused by dThd or dGuo, suggesting that inhibition of cytidine 5'-diphosphate reduction by ribonucleotide reductase is not the only mechanism whereby these nucleosides inhibit leukemic cell cloning. These data suggest that, in addition to inhibiting de novo dCTP production via ribonucleotide reductase, these nucleosides may affect other processes in the salvage pathway such as cellular uptake and phosphorylation or the DNA polymerase reaction itself.

Loss of fluorescence by anthracycline antibiotics: effects of xanthine oxidase and identification of the nonfluorescent metabolites.

Rat liver cytosol and buttermilk xanthine oxidase both converted 7-deoxypyrromycinone, the 7-deoxyaglycone of marcellomycin, a new anthracycline antibiotic, to a nonfluorescent compound under anaerobic conditions and in the presence of an electron donor. Reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate were equally effective electron donors for liver cytosol, and xanthine was the best cofactor for xanthine oxidase. However, xanthine was inactive with liver cytosol. Reactions with xanthine oxidase obeyed Menten-Michaelis kinetics and were inhibited by allopurinol. No xanthine oxidase activity was detected in liver cytosol. Xanthine oxidase also induced a loss of fluorescence when incubated with 7-deoxydaunorubicin aglycone. The nonfluorescent metabolite of 7-deoxypyrromycinone was tentatively identified as the dihydroquinonic derivative of the parent deoxyaglycone on the basis of its spectrophotometric, fluorescent, thin layer chromatographic, and mass spectral characteristics. Our data demonstrate that more than one enzymatic activity, xanthine oxidase, and an unidentified rat liver cytosolic enzyme convert the 7-deoxyaglycones of anthracycline antibiotics to nonfluorescent metabolites.

Comparative anthracycline metabolism in rats: loss of marcellomycin fluorescence.

The in vitro metabolism of marcellomycin by rat tissue fractions showed conversion of marcellomycin to 7-deoxypyrromycinone, bisanhydropyrromycinone, and an as yet unidentified compound by rat liver homogenate, microsomes, cytosol, and mitochondria, and purified hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase, under anaerobic conditions and in the presence of reduced nicotinamide adenine dinucleotide phosphate. All these fractions except the purified reductase subsequently induced a progressive loss of fluorescence. Mitochondria, however, were much less active than microsomes, cytosol, and homogenate in inducing this latter phenomenon. Marcellomycin was converted to 7-deoxyaglycones only partially by nuclei. No loss of fluorescence was observed with this subcellular fraction. No loss of fluorescence was observed when doxorubicin or daunorubicin were incubated under similar conditions. The appearance of a compound with distinct spectrophotometric properties was demonstrated by absorbance spectrometry. The formation of a compound with different fluorescent characteristics was excluded, as was the binding of the aglycones to subcellular components. The activity inducing the loss of fluorescence was studied in greater detail with cytosol. It predominated in the liver and required both an electron donor and anaerobic conditions. The optimal pH for the reaction was between 7.5 and 8.0. Our results suggest the existence of an enzymatic pathway capable of converting the fluorescent nucleus of marcellomycin to a nonfluorescent metabolite.

Schedule-dependent enhancement of 1-beta-D-arabinofuranosylcytosine incorporation into HL-60 DNA by deoxyguanosine.

We studied the ability of the purine deoxynucleoside deoxyguanosine (dGuo) to enhance 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA of HL-60 cultured human leukemia cells. The effects of dGuo on ara-C incorporation into DNA were compared to those of thymidine (dThd), a pyrimidine deoxynucleoside known to augment ara-C effects both in vitro and in vivo. Both deoxynucleosides doubled the cells in the S phase of the cell cycle within the exposure periods (up to 48 hr) and concentrations (10 to 1000 microM) tested. Both deoxynucleosides enhanced ara-C incorporation into DNA equally. However, dThd and dGuo differed in the schedule required to achieve this effect. Simultaneous exposure of cells to ara-C and dThd increased ara-C incorporation into DNA approximately 3.5-fold. Preincubation of cells with dThd for 16 hr prior to the addition of ara-C further enhanced ara-C incorporation into DNA (to approximately 5-fold) in direct proportion to the dThd-induced increase in cells in S phase. Preincubation was essential for dGuo, since 16-hr preincubation of cells with concentrations as low as 30 microM caused augmentation of ara-C incorporation into DNA; but simultaneous exposure of cells to dGuo and ara-C caused no augmentation of ara-C incorporation into DNA. The augmentation of ara-C incorporation into DNA caused by preincubation of the cells with dGuo results from a number of factors, including the cytokinetic effect of increasing the percentage of cells in S phase and the reduction of intracellular dCTP pools. Maximal dGuo enhancement of ara-C incorporation into DNA (approximately 5-fold) required greater than 100 microM dGuo, 16-hr preincubation with dGuo, and final incubation of cells with ara-C after removal of dGuo. We explain this further augmentation of ara-C incorporation into DNA caused by the removal of dGuo prior to adding ara-C by our observed inhibition of ara-C phosphorylation by dGuo concentrations greater than 100 microM.

Inhibitory effects of phytohemagglutinin isolectins L4 and E4 on L1210 cells.

Phytohemagglutinin isolectins L4 and E4 inhibit the growth and proliferation of cultured L1210 murine leukemia cells. L1210 cells were incubated with L4 or E4, and the metabolic and morphological characteristics of the cells were assessed. Dose-dependent inhibition of up to 90% occurs for [3H]thymidine and [14C]uridine incorporation. L4 is 30 to 50 times more potent an inhibitor than is E4. Inhibition begins 2 to 3 hr after exposure of L1210 cells to L4 and persists for as long as the cells are exposed to this isoleuctin. Total DNA and oxygen consumption in L4-treated cultures is also decreased. Whereas protein synthesis assessed by [14C]valine incorporation is less affected, glucose utilization remains unchanged. The binding of L4 and E4 to L1210 cells and human lymphocytes is similar and is reversible by porcine thyroglobulin. Porcine thyroglobulin also reverses L4-induced inhibition of nucleotide incorporation. Cell aggregation is the major morphological consequence of isoleuctin treatment observed by light or electron microscopy. L1210 cells are agglutinated at lower doses of isoleuctins than are normal murine lymphocytes. No evidence of cell death as estimated by 51Cr release or trypan blue uptake has been noted. Our data indicate that L4 and E4 have cytostatic properties and demonstrate that the reversible binding of a macromolecule to the surface of a malignant cell can modulate synthetic pathways and the rate of proliferation.

Cellular accumulation and disposition of aclacinomycin A.

The cellular accumulation and disposition of the anthracycline antitumor antibiotic aclacinomycin A (ACM) were compared to those of daunorubicin. Although both drugs were avidly accumulated by cells, intracellular concentrations of ACM were two to three times those of daunorubicin. Whereas lowered temperature (0 degrees) reduced intracellular accumulation of both drugs, 10 mM sodium azide had no effect on accumulation of either ACM or daunorubicin. Both drugs exited from cells placed in drug-free medium, a process that was reduced at 0 degrees but not altered by 10 mM sodium azide. Unlike whole cells, isolated nuclei accumulated more daunorubicin than ACM. This process was not altered at 0 degrees. Both drugs were lost from nuclei placed in drug-free buffer, a process that was reduced at 0 degrees. Unlike daunorubicin, which localized in cell nuclei, ACM localized in the cytoplasm with no detectable nuclear fluorescence. Although both drugs produced dose-dependent inhibitions of [3H]thymidine and [3H]uridine incorporation by L1210 and P388 cells, ACM inhibited both processes at lower concentrations than did daunorubicin. While daunorubicin inhibited [3H]thymidine incorporation more effectively than [3H]uridine incorporation, the reverse was observed with ACM.

Cellular pharmacology in murine and human leukemic cell lines of diaziquone (NSC 182986).

We investigated the in vitro interaction with and antitumor effect on several murine and human leukemic cell lines of diaziquone (AZQ). L1210 cells accumulated AZQ from Roswell Park Memorial Institute Medium 1640 with or without newborn calf serum by a temperature-dependent and sodium azide-resistant process. AZQ inhibited, in a dose-dependent fashion, [3H]thymidine incorporation into L1210 cells, but this inhibition was slow to develop, requiring approximately 6 hr to become apparent. The minimal inhibitory concentration of AZQ for this process was 0.05 to 0.25 nmol/ml. AZQ was a much less effective inhibitor of L1210 cell [3H]uridine and [14C]valine incorporation. In suspension cultures, AZQ inhibited growth of L1210 and HL-60 cells at minimal inhibitory concentrations of 0.5 to 1 nmol/ml. In soft agar cultures, AZQ inhibited HL-60 cell cloning at minimal inhibitory concentrations of 0.1 to 0.3 nmol/ml. AZQ provoked a dose-dependent increase in oxygen consumption when added to intact L1210, HL-60, and K562 cells and was converted to an AZQ anion free radical by these cells. When the aziridine rings of AZQ were opened by acid treatment, the resulting molecule was not accumulated by L1210 cells, did not provoke O2 consumption, did not form free radicals when added to L1210 cells, and was a much less effective inhibitor of [3H]thymidine incorporation by L1210 cells than was AZQ.

Murine metabolism and disposition of iron:adriamycin complexes.

Previous studies have reported antitumor activity and reduced cardiotoxicity for a putative 3:1 complex of iron:Adriamycin (ADR). We have studied the tissue distribution and metabolism of a wide variety of freshly prepared and lyophilized iron:ADR preparations after administration to BALB/c mice (ADR, 16 mg/kg i.v.). Tissue concentrations of ADR given without iron were initially highest in kidney and liver, and ADR fluorescence was lost from all tissues except the spleen with a t1/2 of 15 to 18 hr. ADR remained the major fluorescent species in liver and kidney from 0.5 to 72 hr after treatment. Freshly prepared iron:ADR (1:1) behaved similarly to ADR except for a slightly longer tissue t1/2 in heart, liver, and kidney. The tissue distribution of freshly prepared 2:1 and 3:1 iron:ADR was very different from that of ADR without iron; lung containing the highest concentrations of ADR fluorescence. Administration of freshly prepared 1:1, 2:1, and 3:1 iron:ADR resulted in some increase in adriamycinol in the liver, but ADR was always the major fluorescent species present. The tissue distribution of 1:1 iron:ADR that had been aged for 48 or 96 hr was similar to that of fresh 2:1 and 3:1 iron:ADR rather than ADR or fresh 1:1 iron:ADR. When lyophilized iron:ADR preparations were reconstituted and administered, the 0.5-hr tissue distribution of 0.1:1, 0.2:1, 0.25:1, and 0.33:1 iron:ADR was the same as ADR alone, but 0.5:1, 1:1, 2:1, and 3:1 iron:ADR were all accumulated primarily in the lung. Physicochemical studies confirm the production of microaggregated iron:ADR complexes and light microscopy allows visualization and sizing of these aggregates. We feel that trapping of these iron:ADR aggregates in the pulmonary vascular bed accounts for the observed dramatic alteration in tissue distribution. Light and electron microscopic studies confirm the intravascular sequestration of iron in pulmonary capillaries.

Growth inhibition by thymidine of leukemic HL-60 and normal human myeloid progenitor cells.

We compared the relative susceptibility of HL-60, a human acute progranulocytic leukemia cell line, and the normal human myeloid progenitor cell, the colony-forming unit in culture, to growth inhibition by thymidine. Normal human myeloid colony formation was more sensitive to thymidine than was HL-60 colony formation, the former being inhibited by greater than or equal to 0.5 mM thymidine and the latter by greater than or equal to 5.0 mM. Colony inhibition by thymidine was irreversible in both cases after a seven-day incubation of the colony-forming unit in culture in liquid medium enriched with thymidine and subsequent replating in thymidine-free soft agar. Thymidine-induced inhibition of HL-60 cloning could be partially prevented by deoxycytidine, whereas normal myeloid cloning could not, suggesting that high-concentration thymidine may affect processes necessary for cloning in addition to deoxyribonucleotide synthesis. HL-60 growth in liquid suspension culture could be suppressed transiently by 1.0 mM thymidine, suppressed totally by greater than or equal to 5.0 mM thymidine, and rescued completely by concomitant addition of deoxycytidine. The development of resistance to 1.0 mM thymidine could not be accounted for by reduction in thymidine pool size or by reduction in thymidine kinase activity. Replating of HL-60 cells growing in the presence of 1.0 mM thymidine in liquid medium in thymidine-free soft agar produced significant colony inhibition, also suggesting that thymidine affects more than just deoxyribonucleotide synthesis or that the clonogenic HL-60 cell presents a distinct subpopulation with more sensitive deoxyribonucleotide-synthetic control mechanisms.

Free radicals in quinone containing antitumor agents. The nature of the diaziquone (3,6,-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone) free radical.

The enzymatically generated free radical of the antitumor agent diaziquone is analyzed with the help of two analogs where either the aziridine rings (RQ14) or the carboethoxyamino groups (RQ2) were substituted by chlorine atoms. The hyperfine couplings observed in the diaziquone free radical are due to the nitrogens in the aziridine group. Unresolved coupling and hindered rotation contribute to line broadening. We find that diaziquone free radicals are more stable than RQ14 but less stable than RQ2 free radicals. The reason for this is that the carboethoxyamino groups make the aromatic ring unstable, while the aziridines contribute to its stability. The free radical observed in diaziquone is in all probability that of the parent compound and not that of an intermediate metabolite.

Purification of the phytohemagglutinin family of proteins from red kidney beans (Phaseolus vulgaris) by affinity chromatography.

Half-gram quantities of phytohemagglutinin lectins are purified from saline extracts of red kidney beans (Phaseolus vulgaris) by affinity absorption on porcine thyroglobulin-Sepharose. All of the mitogenic and erythroagglutinin activity of the saline extract is removed by this absorbent, and 74% of the original erythroagglutinating activity elutes from the affinity absorbent representing a 25-fold purification. Five distinct proteins appear in the polyacrylamide gel electrophoresis of the affinity absorbent eluate. Although all five proteins specifically bind to porcine thyroglobulin, the cathodal migrating proteins bind more strongly than the anodal migrating proteins. The most cathodal proteins are potent erythroagglutinins. This simple, efficient method is used to prepare all the active components of the phytohemagglutinin family in large yield and high purity.

Phytohemagglutinin isolectin subunit composition.

The subunit compositions of individual phytohemagglutinin isolectins from red kidney bean Phaseolus vulgaris were examined by isoelectric focusing and sodium dodecyl sulfate electrophoresis on polyacrylamide gels. Isoelectric focusing reveals heterogeneous but unique and non-overlapping protein band patterns for each of the homotetrameric isolectins, E4 and L4. Isoelectric focusing of the intermediate isolectins which contain both subunits (E3L1, E2L2, and E1L3) show all the protein bands common to isolectins E4 or L4 in proportions relative to their suggested subunit compositions. Polyacrylamide gel electrophoresis in a continuous sodium dodecyl sulfate buffer system gives a single protein band for all of the isolectins. In contrast, a discontinuous sodium dodecyl sulfate buffer procedure resolves isolectins E4 and L4 into single major protein bands of apparent molecular weights 31 700 (+/-600) and 29 900 (+/-200), respectively. Each of the intermediate isolectins contained both protein bands and their relative proportion, as determined by absorbance scanning, confirms the phytohemagglutinin isolectin subunit compositions as E4, E3L1, E2L2, E1L3, and L4.