Concanavalin a promotes the uptake of lysosomal hydrolases by human fibroblasts

Human placental hexosaminidase B and β-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4°C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37°C is refractory to haptene treatment. These results are interpreted as an initial binding of enzyme to concanavalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Physicochemical characterization of human fibroblast migration inhibitory factor

We recently reported a new lymphokine activity that affects fibroblasts by inhibiting their spontaneous migration. Human fibroblast migration inhibitory factor (FIF) obtained from concanavalin A (Con A)-stimulated human lymphocytes was characterized by Sephadex gel filtration and by enzyme treatment. FIF was found to be stable at 56 degrees C for 15 min but destroyed at 80 degrees C or at pH lower than 5. Gel filtration revealed two peaks of FIF activity 15,000 and at 34,000 Da. FIF activity was lost following treatment with trypsin, chymotrypsin, and neuraminidase and FIF could not be generated in the presence of inhibitors of glycosylation, suggesting that the molecule was a glycoprotein. FIF could be removed by adsorption to human fibroblasts but not to PMN, monocytes, or red blood cells. Further studies were carried out to investigate the role of sugars in the interaction of FIF with the target cells. Human FIF activity was significantly reduced in the presence of several sugars including alpha-methyl-D-mannoside, L-xylose, N-acetyl-D-glucosamine, D-mannose, L-rhamnose but not L-fucose. Preincubation of human fibroblasts with alpha-methyl-D-mannoside prevented their response to FIF. In contrast, pretreatment of fibroblasts with mannosidase had no effect, suggesting that alpha-methyl-D-mannoside was an essential component of the FIF molecule recognized by the FIF receptor on fibroblasts.     

Interactions of concanavalin A with chick embryo fibroblasts transformed by Rous sarcoma virus. Study with an RSV mutant thermosensitive for transformation.

The interactions between concanavalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 degrees C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 degrees C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 degrees C.     

Type I procollagen N-proteinase from whole chick embryos. Cleavage of a homotrimer of pro-alpha 1(I) chains and the requirement for procollagen with a triple-helical conformation

Procollagen N-proteinase, the enzyme which cleaves the NH2-terminal propeptides from type I procollagen, was purified over 15,000-fold from extracts of chick embryos by chromatography on columns of DEAE-cellulose, concanavalin A-agarose, heparin-agarose, pN-collagen-agarose, and a filtration gel. The purified enzyme had an apparent molecular weight of 320,000 as estimated by gel filtration and a pH optimum for activity of 7.4 to 9.0. The enzyme was inhibited by metal chelators and the thiol reagent dithiothreitol. Addition of calcium was required for maximal activity under the standard assay conditions, and the presence of calcium decreased thermal inactivation at 37 degrees C. The purified enzyme cleaved a homotrimer of pro-alpha 1(I) chains, an observation which indicated that the presence of pro-alpha 2(I) chain is not essential for the enzymic cleavage of NH2-terminal propeptides. Previous observations suggesting that the enzyme requires a substrate with a native conformation were explored further by reacting the enzyme with type I procollagen at different temperatures. Type I procollagen from chick embryo fibroblasts became resistant to cleavage at about 43 degrees C. Type I procollagen from human skin fibroblasts, which was previously shown to have a slightly lower thermal stability than chick embryo type I procollagen, became resistant to cleavage at temperatures that were about 2 degrees C lower. The results suggested that the enzyme is a sensitive probe for the three-dimensional structure of the NH2-terminal region of the procollagen molecule and that it requires the protein substrate to be triple helical.     

Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.     

Subcellular distribution and movement of 5''-nucleotidase in rat cells.

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.     

ConA-mediated binding and uptake of purified alpha-galactosidase A in Fabry fibroblasts

In most human tissues there are at least two different alpha-galactosidases, A and B. The former is deficient in patients hemizygous for Fabry disease. We have isolated it from human placenta and found that it was labile even at culture conditions, but was stabilized after binding to concanavalin A (conA). The alpha-galactosidase activity was markedly increased in Fabry fibroblasts when these were treated with conA and exposed to alpha-galA at 37 degrees C. The maximum activity was obtained after 1/2-2 h of incubation and was maintained for at least 4 h. The binding and uptake of conA into Fabry cells was followed by microscopical studies of fluorescein-labelled conA. We assume that alpha-galA is taken up by endocytosis of the enzyme-conA complex.     

Dynamic state of concanavalin A receptor interactions on fibroblast surfaces.

Cultured normal and transformed fibroblasts were treated "in situ" by the concanavalin A-peroxidase labelling technique. It is known that peroxidase recognizes only a fraction of the bound lectin depending on the cell type. Kinetics studies revealed that 80 to 95 percent of the peroxidase and only 10 percent of the lectin are released from the cell surface when the labelled cells were reincubated at 37 degrees C. It is shown that it is mostly the concanavalin traced by peroxidase that is released and also that the lectin and the enzyme are shed as a complex or concomitantly. Consequently, the shedding pattern of the enzyme is used to demonstrate heterogeneity in the lectin binding sites; there are two main components labelled by concanavalin and peroxidase, one which has a short period (from 6 to 16 min) and another one with a much longer one (1.3 to 3 h). It is shown that when cells are incubated at 37 degrees C after a lectin treatment, secondary binding forces occur between the lectin and cell surface components which render the lectin unavailable for inhibiting sugars. Under the same conditions, some peroxidase can still be bound and a slight agglutination can still occur.     

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.     

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.     

Large-scale isolation of the Neurospora plasma membrane H+-ATPase

A method for the purification of relatively large quantities of the Neurospora crassa plasma membrane proton translocating ATPase is described. Cells of the cell wall-less sl strain of Neurospora grown under O2 to increase cell yields are treated with concanavalin A to stabilize the plasma membrane and homogenized in deoxycholate, and the resulting lysate is centrifuged at 13,500g. The pellet obtained consists almost solely of concanavalin A-stabilized plasma membrane sheets greatly enriched in the H+-ATPase. After removal of the bulk of the concanavalin A by treatment of the sheets with alpha-methylmannoside, the membranes are treated with lysolecithin, which preferentially extracts the H+-ATPase. Purification of the lysolecithin-solubilized ATPase by glycerol density gradient sedimentation yields approximately 50 mg of enzyme that is 91% free of other proteins as judged by quantitative densitometry of Coomassie blue-stained gels. The specific activity of the enzyme at this stage is about 33 mumol of P1 released/min/mg of protein at 30 degrees C. A second glycerol density gradient sedimentation step yields ATPase that is about 97% pure with a specific activity of about 35. For chemical studies or other investigations that do not require catalytically active ATPase, virtually pure enzyme can be prepared by exclusion chromatography of the sodium dodecyl sulfate-disaggregated, gradient-purified ATPase on Sephacryl S-300.     

Cooperativity of the concanavalin A inhibition of bovine milk fat globule membrane 5'-nucleotidase. Response to extraction of nucleotidase and of putative cytoplasmic surface coat components

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) of bovine milk fat globules can be solubilized by deoxycholate from either isolated globule membranes or washed cream. The solubilized and membrane-bound enzymes exhibit similar Km values and are inhibited by concanavalin A by an apparent noncompetitive process. The soluble enzyme shows positive cooperativity for the inhibition (Hill coefficient of 2) at 37 degrees C, but the membrane enzyme exhibits essentially no cooperation effect. At lower temperatures (5 or 20 degrees C) the cooperative effect in the inhibition of the soluble enzyme is lost. Colchicine and cytochalasin D failed to induce cooperativity of the concanavalin A inhibition of the membrane enzyme, but induction cooperativity occurred when membranes were extracted with glycine/EDTA/mercaptoethanol, releasing a major protein component with a polypeptide molecular weight of 155 000. We suggest that the interaction of this component with the membrane imposes restraints on the behavior of the nucleotidase which are reflected in the cooperativity of the inhibition of the enzyme by concanavalin A.     

Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 degrees C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and beta-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+-ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPase activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.     

The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Concanavalin A alters the turnover rate of tyrosine aminotransferase in cultured hepatoma cells

Concanavalin A added to monolayer cultures of Reuber H-35 hepatoma cells caused a rapid inactivation of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, E.C. 2.6.1.5) and loss of reactivity with antibody against the native, dimeric enzyme. Analysis of treated cells with an antibody raised against carboxymethylated, denatured enzyme showed that the inactivated enzyme was reactive with this reagent, which does not react with the native enzyme. Subsequent addition of alpha-methyl-D-mannopyranoside to remove concanavalin A restored both enzyme activity and reactivity to antibody against native enzyme. After long-term treatment with concanavalin A, the restored enzyme levels were significantly higher than in controls treated with the sugar but not the lectin. Analysis of the turnover of the enzyme by two methods revealed that the rate of its degradation is reduced about 2-fold in concanavalin A-treated cells. Treatment with H-35 cells with concanavalin A thus effects an alteration in conformation of tyrosine aminotransferase, rendering it somewhat less sensitive to intracellular degradation.     

Reversibility of monensin inhibition of oligosaccharide processing of human fibronectin

Monensin impairs oligosaccharide processing in fibronectin primarily by inhibiting the conversion of oligosaccharides from the high mannose type to the complex type. The separate effects of monensin and cations on alpha-mannosidase activity in fibroblasts were examined using an in vitro assay system. The results indicated that monensin did not directly inhibit alpha-mannosidase activity in vitro, although prior treatment of fibroblasts with monensin caused an irreversible suppression of enzyme activity. The reversibility of monensin action on oligosaccharide processing was also examined. Analyses using concanavalin A (ConA) Sepharose affinity chromatography showed that the inhibitory action of monensin on oligosaccharide processing was biologically reversible. A progressive return to complex type oligosaccharides began about 11 h after the removal of the monensin. These composite results indicate that the reversibility of monensin action on oligosaccharide processing in fibronectin may be attributed to the restoration of enzyme activity, although the mechanism by which restoration occurs remains to be deciphered.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Phospholipid metabolism of stimulated lymphocytes. Comparison of the activation of acyl-CoA:lysolecithin acyltransferase with the binding of concanavalin A to thymocytes.

Calf thymocytes were isolated and incubated with concanavalin A. The effect of the mitogen on the enzyme activity of membrane-bound lysolecithin acyltransferase (acyl-CoA:1-acylglycero-3-phosphorylcholine-O-acyltransferase, EC 2.3.1.23) was determined as also the binding of 125I-labelled concanavalin A to intact cells and isolated membranes. The lysolecithin acyltransferase was found to be activated three times in microsomal membranes. The activation occurred directly after binding of concanavalin A and was temperature independent, since similar activities were found in cells treated with concanavalin A at 0 and 37 degrees C. The acyltransferase activation using increasing concentrations of concanavalin A revealed a different behaviour, as compared to the binding of concanavalin A. While the binding of concanavalin A to intact cells expressed a normal hyperbolic saturation function the activation process of the acyltransferase described a sigmoidal relationship. Correspondingly, the interaction coefficients for both functions were different (Sips coefficient for binding = 1.0 and Hill coefficient of the enzyme activation = 1.8). These results indicate that the acyltransferase activation is due to a cooperative interaction between the ligand-receptor complex and the enzyme.     

An enzyme activity in normal and ataxia telangiectasia cell lines which is involved in the repair of gamma-irradiation-induced DNA damage.

An enzyme that enhances the activity of DNA polymerase I (EC 2.7.7.7) for gamma-irradiated calf thymus DNA was demonstrated in cellular extracts of normal human fibroblasts and lymphoid-cell lines. This enzyme was found to be deficient in all cellular extracts of fibroblasts and lymphoid-cell lines examined from patients with the autosomal recessive disease ataxia telangiectasia. The activity in cellular extracts from normal fibroblasts was removed when heated to 100 degrees C for 2 min or when the assay was performed at 4 degrees C. No significant deficiency in primer-activating enzyme activity was observed in cell-free extracts of lymphoid lines from patients with xeroderma pigmentosum, Huntington's chorea or neurofibromatosis, or from an ataxia telangiectasia heterozygote.