Increased surface binding sites of insulin in ML236B (compactin)-resistant mutants of Chinese hamster cell line

Mutants resistant to ML236B (compactin) were isolated from the Chinese hamster lung V79 cell line (1). Three ML236B-resistant mutants, MF-1, MF-2 and MF-3, were enhanced in insulin-specific binding activity about 2 to 3 times over the parental V79 cell lines. Compared to V79, endocytosis of insulin was also increased 2 to 3-fold in ML236B-resistant mutants than V79. Scatchard analysis showed that 5,000 insulin binding sites per cell in V79 and 16,000 in a NL236B-resistant clone, MF-2. Insulin receptors in mutant and parental strains are down-regulated to a similar extent in the parental V79 treated with an excess insulin. This is the first somatic cell mutant with increased surface binding sites for insulin.     

Chinese hamster cell mutants with defective endocytosis of low-density lipoprotein contain altered fatty acid composition in the membrane

Chinese hamster V79 cell mutants resistant to compactin (ML236B), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, are defective in endocytosis of low-density lipoprotein (1). Two resistant clones, MF-2 and MF-3, differ in lipid composition from the parental V79 strain. In both the total cells and membrane fraction, the ratio of palmitoleic acid (16:1)/palmitic acid (16:0) is 0.4-0.5 in MF-2 and 1.7-1.8 in MF-3 while that in V79 is 0.2-0.3. By contrast, a hybrid clone between V79 and MF-3 shows a ratio of palmitoleic acid to palmitic acid very similar to that of V79. The synthesis of palmitoleic acid from acetate in the resistant clone is higher than in V79.     

Ultrastructural changes during the enhancement of cellular 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase in a Chinese hamster cell mutant resistant to compactin (ML 236B)

Chinese hamster V79 cell mutants resistant to compactin (ML236B) were isolated. A resistant clone, MF-2, grown in the presence of 2 micrograms/ml of ML236B for 1 week showed a 30-fold increase in 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-Co A) reductase activity compared to MF-2 grown in the absence of ML236B, and cells grown for 4 weeks showed a 53-fold increase. Apparent ultrastructural changes in thin sections of the MF-2 cells were observed after growth in ML236B: dilated cisternae in the rough endoplasmic reticulum had or did not have flocculated contents; there was significant distension of perinuclear space; and vesicular inclusion bodies were present in nuclei.     

Altered function and structure of low-density lipoprotein receptor in compactin (ML236B)-resistant mutants of Chinese hamster cells

Mutants resistant to compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have been previously isolated from the Chinese hamster V79 cell line. Two compactin-resistant mutants, MF-1 and MF-2, show altered responses to human low-density lipoprotein (LDL). Accumulation of fluorescent-labeled LDL was much reduced. Ligand blotting showed LDL receptor activity in MF-1 and MF-2 cells of about one half to one third that of V79. Internalization and degradation of LDL in MF-1 or MF-2 cells were about one tenth those in V79 cells, suggesting that the LDL binding as well as the LDL internalization of the compactin-resistant clones was altered. Down-regulation of LDL receptor activity as well as hydroxymethylglutaryl CoA reductase was observed in V79 cells treated with LDL, while there appeared to be much less down-regulation in MF-1 and MF-2 cells. Using anti-LDL receptor antibody, MF-1 and MF-2 cells were found to produce smaller-sized mature forms of LDL receptor: the molecular mass of the mutant LDL receptor was 3-5 kDa smaller than that of the parental LDL receptor. Altered O-linked oligosaccharides or amino acid sequence might account for the decreased molecular mass and aberrant properties of the LDL receptor in MF-1 and MF-2.     

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.     

Secondary mutation resistant to 7-ketocholesterol rescues a sterol metabolic defect in amphotericin B-resistant Chinese hamster cell line

Amphotericin B-resistant mutants isolated from Chinese hamster V79 cells (1) are defective in cholesterol synthesis and more sensitive to an oxygenated sterol analog, 7-ketocholesterol, than their parental cell line. We isolated 7-ketocholesterol-resistant mutants from an amphotericin B-resistant mutant, AMBR-1. The 7-ketocholesterol-resistant mutants had regained increased level of free cholesterol, and they showed somewhat similar dose-response curves to amphotericin B as that of V79. Sterol synthesis from acetate, but not from mevalonate, in 7-ketocholesterol-resistant clones was threefold higher than that of AMBR-1. 7-Ketocholesterol-resistant clone, unlike AMBR-1, could form colonies in the presence of lipoprotein-depleted serum. The results are discussed in terms of probable change in the sterol biosynthetic pathway by the different lesions.     

Inhibition of cholesterol synthesis and esterification regulates high density lipoprotein interaction with isolated epithelial cells of human small intestine

The effect of two inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lovastatin and monacolin L, and an inhibitor of acyl coenzyme A:cholesterol acyltransferase (ACAT), Sandoz compound 58-035, on the interaction of 125I-labeled high density lipoprotein-3 (HDL3) with isolated human enterocytes was studied. Both HMG-CoA reductase inhibitors inhibited cholesterol synthesis and 125I-labeled HDL3 binding and degradation by enterocytes; a strong correlation between changes in cholesterol synthesis and interaction of 125I-labeled HDL3 with cells was observed. Lovastatin caused reduction of the apparent number of 125I-labeled HDL3 binding sites without affecting the binding affinity. No changes of cell cholesterol content were observed after incubation of cells with lovastatin. Mevalonic acid reversed the effect of lovastatin on 125I-labeled HDL3 binding. Lovastatin blocked up-regulation of the HDL receptor in response to loading of cells with nonlipoprotein cholesterol and modified cholesterol-induced changes of 125I-labeled HDL3 degradation. Lovastatin also reduced HDL-mediated efflux of endogenously synthesized cholesterol from enterocytes. The ACAT inhibitor caused a modest increase of 125I-labeled HDL3 binding to enterocytes and significantly decreased its degradation; both effects correlated with inhibition of cholesteryl ester synthesis. The results allow us to assume that the intracellular free cholesterol pool may play a key role in regulation of the HDL receptor.     

Isolation and cross-sensitivity of X-ray-sensitive mutants of V79-4 hamster cells

The V79-4 Chinese hamster line was mutagenized and surviving clones screened for X-ray sensitivity using a replica microwell technique. One slightly sensitive clone and 3 clearly sensitive clones were isolated from approximately 5000 screened, and designated irs 1 to irs 4. The 3 more sensitive clones showed different responses to the genotoxic agents mitomycin C (MMC), ethyl methanesulphonate (EMS) and ultraviolet light (UV). irs 1 showed considerable sensitivity to all the agents tested, in the order MMC much greater than EMS greater than UV. irs 2 and irs 3 had similar sensitivities to EMS and to UV (EMS greater than UV) but irs 3 was more sensitive than irs 2 to MMC. None of these mutants is identical in phenotype to previously published mutants.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant Chinese hamster ovary cell line

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (CR-7), and revertant (RCR-7) cells. In contrast to WT and RCR-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2-14C]mevalonolactone revealed that CR-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but CR-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that CR-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1-2 micrograms/ml) strongly and reversibly suppressed reductase activity in WT and RCR-7. CR-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.     

Molecular analysis of X-ray-induced mutants at the HPRT locus in V79 Chinese hamster cells

Spontaneous and X-ray-induced mutants at the hypoxanthine phosphoribosyl transferase (HPRT) locus have been isolated from V79 Chinese hamster cells and characterized at the biochemical and cytogenetic levels. Fourteen spontaneous and 24 X-ray-induced clones were azaguanine and thioguanine resistant, did not grow in HAT medium (AZRTGRHATS) and failed to incorporate significant levels of [14C]hypoxyanthine. Cytogenetic analysis of two spontaneous and eight X-ray-induced mutants revealed no major X chromosome rearrangements. In two induced mutants, one of which was hypotetraploid (mode 35-39) with 2 X chromosomes, the short arm of the chromosome (Xp) was slightly shorter than normal. A third mutant was hyperdiploid (mode 22-23) compared with the parental clone (mode 21). When compared with wild-type clones, no other cytogenetic changes were evident in the remaining mutants. Analysis at the DNA level using a Chinese hamster HPRT cDNA probe showed major deletion of HPRT sequences in two and partial deletion in another two induced mutants. In two of the mutants with deletions of HPRT sequences there was a visible shortening of the Xp arm. In the other six mutants two spontaneous and four induced) no karyotypic changes or alterations in restriction fragment patterns were detected suggesting that they carry small deletions or point mutations at the HPRT locus.     

Application of computer to monitoring and control of fermentation process: Microbial conversion of ML-236B Na to pravastatin

An automatic feeding process for microbial hydroxylation of ML236B sodium salt (ML-236B Na; compactin) by Streptomyces carbophilus SANK 62585 was developed. The hydroxylated product, pravastatin sodium salt (pravastatin; trade name Mevalotin), is an inhibitor of 3-hydroxy-3-methyglutaryl-coenzyme A reductase (HMG-CoA reductase) used as cholesterol-lowering drug. The hydroxylation activity of S. carbophilus was induced by the addition of ML236B Na to culture broth but inhibited by high concentration of ML236B Na. In order to obtain high conversion yield, it was necessary to maintain optimum ML236B Na concentration throughout the fermentation by continuous feeding. For this purpose, we developed an on-line monitoring method, which mainly consisted of a cross-flow filtration module, high-performance liquid chromatography (HPLC) analyzer, feed pump, and microcomputer for regulation of ML236B Na concentration. An algorithm for control of ML236B Na feed rate based on feedback and feed-forward control where conversion rate after Deltat was estimated by using regression analysis of the five latest values of conversion rate. In a fed-batch culture employing this system, the concentration of ML236B Na was maintained at optimum level during the fermentation and the productivity of pravastatin was increased threefold over that obtained in manual control culture. (c) 1993 John Wiley & Sons, Inc.     

The use of biotin-streptavidin systems for enhancing the parameters of immunometric analysis

The possibility of improving analytical parameters of the immunometric assay with the use of biotinylated antibodies and biotin-streptavidin complexes in comparison with the commonly known approach of direct antibody modification with 125I has been studied. Experiments have been carried out with the use of low-affinity antibodies (Kass approximately 10(9) M-1) to ferritin. The signal-to-noise ratio in the immunometric increases 2.3 times when streptavidin labeled with horse-radish peroxidase is used and 4.3 times when the preformed streptavidin + biotin-peroxidase complex is used in comparison with assay systems based on 125I-labeled antibodies. The improvement of assay parameters of immunochemical systems by means of biotin-streptavidin complexes has been found to permit the use of low-affinity antibodies as assay reagents, thus ensuring analytical parameters attaining or close to those of immunoradiometric assay systems based on high-affinity 125I-labeled antibodies (Kass approximately 10(10) M-1). As shown in this study, the following factors ensure the signal enhancement in biotin-streptoavidin systems: (a) the biotin modification of several lysin residues per IgG molecule, the optimum extent of modification being 3-4 residues per molecule; (b) mild procedure for biotinylation. In contrast to oxidative iodination, the modification of NH2 groups with biotin esters does not significantly affect their antigen-binding properties.     

Receptors for modified low-density lipoproteins on human endothelial cells: different recognition for acetylated low-density lipoprotein and oxidized low-density lipoprotein

We examined the uptake pathway of acetylated low-density lipoprotein and oxidatively modified LDL (oxidized LDL) in human umbilical vein endothelial cells in culture. Proteolytic degradation of 125I-labeled Ac-LDL or Ox-LDL in the confluent monolayer of human endothelial cells was time-dependent and showed saturation kinetics in the dose-response relationship, which suggests that their incorporation is receptor-mediated. Cross-competition studies between acetylated LDL and oxidized LDL showed that the degradation of 125I-labeled acetylated LDL was almost completely inhibited by excess amount of unlabeled acetylated LDL, while only partially inhibited by excess unlabeled oxidized LDL. On the other hand, the degradation of 125I-labeled oxidized LDL was equally inhibited by excess amount of either acetylated or oxidized LDL. Cross-competition results of the cell-association assay paralleled the results shown in the degradation assay. These data indicate that human endothelial cells do not have any additional receptors specific only for oxidized LDL. On the contrary, they may have additional receptors, as we previously indicated on mouse macrophages, which recognize acetylated LDL, but not oxidized LDL.     

Membrane receptors on rat hepatocytes for the inner core region of bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) are potent endotoxins that are thought to be involved in the pathogenesis of Gram-negative septicemia. The liver is known to be the primary organ responsible for the clearance of LPS from the systemic circulation in mammals. In this work, 125I-labeled LPS have been used in a filtration assay for the specific binding of LPS to intact rat hepatocytes. Eight S-form (smooth) LPS with complete O-specific polysaccharide chains isolated from different O-serotypes of Salmonella and Escherichia coli as well as nine R-form (rough) LPS isolated from Salmonella mutants deficient in synthesis of their core oligosaccharides were used in this study. All 125I-labeled S-form LPS and R-form LPS, except Re, show specific binding to isolated hepatocytes. The binding is saturable, is inhibited with excess unlabeled homologous or heterologous LPS but not lipid A, and is trypsin sensitive. L-Glycero-D-mannoheptose (heptose), a constituent of the inner core region of almost all LPS, is a potent inhibitor of the specific binding of 125I-labeled Rb2 LPS, whereas other monosaccharides, including 3-deoxy-D-manno-2-octulosonic acid (KDO), have weak or negligible inhibitor activity. These results strongly suggest the presence of a lectin-like receptor for the LPS inner core region (heptose-KDO region) on the plasma membrane of rat hepatocytes.     

Sensitivity to DNA-damaging agents and mutation induction by UV light in UV-sensitive CHO cells

Three UV-sensitive (UVs) mutants isolated from a CHO cell line were analyzed for survival after exposure to H2O2, EMS, MMC, CCNU, X-rays and for mutation induction after UV-irradiation. The UVs mutants showed normal sensitivities to EMS and H2O2, whereas they were hypersensitive to the bifunctional alkylating agents MMC and CCNU and to hypoxic X-irradiation. Compared to parental cells, one of the UV-sensitive clones showed approximately 3- and 7-fold enhancement in the mutagenic response per unit UV dose for 6-thioguanine and ouabain resistance, respectively.     

Biological and biochemical characterisation of purine analogue resistant clones of V79 Chinese hamster cells

Purine analogue resistant clones have been selected from the closely related Chinese hamster lines V79A and V79S. Clones were of either spontaneous origin or induced by EMS or ultraviolet light. The majority of clones selected in 8-azaguanine showed stable cross resistance to 6-thioguanine. Clones derived from V79A and selected for 6-thioguanine resistance were cross resistant to 8-azaguanine: however a group of 6-thioguanine resistant mutants selected from V79S cells were 8-azaguanine sensitive. All clones except two were unable to grow in HAT medium. The two exceptions were 8-azaguanine resistant, showed partial sensitivity to 6-thioguanine, and also differed in other biochemical characteristics. HGPRT activity was measurable in extracts of all clones under standard conditions. In many clones, HGPRT activity increased as the hypoxanthine concentration was reduced. Whole cell uptake of [14C] hypoxanthine was low in all cases examined and was not modified by incubation in the presence of amethopterin. The heat sensitivity and electrophoretic mobility of HGPRT in extracts of some clones was compared to that in wild-type extracts. All clones tested except one, which was consistently HAT positive, showed enhanced heat sensitivity and reduced electrophoretic mobility. None of the mutants reverted spontaneously at detectable frequency but some could be induced to revert by EMS. The presence of measurable enzyme with altered properties in all clones suggests that these revertable drug resistant clones represent missense mutants.     

The gene for ornithine decarboxylase is co-amplified in hydroxyurea-resistant hamster cells

We have constructed a cDNA library from the highly hydroxyurea-resistant hamster cell line 600H in which the activity of ribonucleotide reductase is elevated more than 80-fold. Using the technique of differential hybridization, we have isolated a number of cDNA clones from this library which are homologous to genomic DNA sequences amplified in the 600H cell line compared to the V79 parental line. One of these cDNA clones by sequence analysis was found to code for ornithine decarboxylase. This was confirmed by in vitro translation of poly(A+) RNA isolated by hybridization-selection followed by immunoprecipitation with antiserum specific for mouse ornithine decarboxylase. Genomic sequences homologous to the cDNA clone were shown to be sequentially amplified 6-20-fold in hamster cell lines selected stepwise for resistance to increasing concentrations of hydroxyurea. Genomic sequences homologous to a cDNA for the M2 subunit of ribonucleotide reductase were also amplified in these cell lines, and the degree of M2 sequence amplification corresponded to the degree of amplification of ornithine decarboxylase sequences, suggesting that the two genes had been co-amplified during the selection of the hydroxyurea-resistant phenotype.     

Radioimmunoassay of rat apolipoprotein B peptides in lipoproteins and tissues

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) separates rat apolipoprotein B (apoB) into one lower and two higher molecular weight components. Of the latter, peptide I (PI) corresponds to human B-100, while the slightly faster-migrating peptide II (PII) lacks a human counterpart; the smaller species peptide III (PIII) corresponds to human B-48. We describe here a competitive radioimmunoassay which separately measures the amounts of total (i.e., PI + PII + PIII) and larger (i.e., PI + PII) rat apoB peptides, with the amounts of PIII obtained by difference. Standard rat PIII and combined PI + PII (PI,II) were isolated by high-pressure gel filtration liquid chromatography in the presence of SDS, and the PI,II was used as an immunogen to raise rabbit antisera which were capable of binding all three forms of rat apoB. However, Scatchard analysis showed this binding to represent two distinct types of antibodies: one high-affinity class which bound only PI,II and a second class which bound all apoB peptides with equal but lower affinity. Thus, since 125I-labeled PIII was displaced equally effectively by PI,II and PIII, but 125I-labeled PI,II was displaced only by PI,II, the unabsorbed antiserum could be used to measure either total apoB or PI,II alone, depending on the choice of labeled ligand. The validity of the assay for apoB peptides in very-low-density and low-density lipoproteins and in liver microsomes was verified by comparison with peptide determinations by SDS-PAGE.     

Differences in the processing of chylomicron remnants and beta-VLDL by macrophages

To gain a detailed understanding of those factors that govern the processing of dietary-derived lipoprotein remnants by macrophages we examined the uptake and degradation of rat triacylglycerol-rich chylomicron remnants and rat cholesterol-rich beta-very low density lipoprotein (beta-VLDL) by J774 cells and primary cultures of mouse peritoneal macrophages. The level of cell associated 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants reached a similar equilibrium level within 2 h of incubation at 37 degrees C. However, the degradation of 125I-labeled beta-VLDL was two to three times greater than the degradation of 125I-labeled chylomicron remnants at each time point examined, with rates of degradation of 161.0 +/- 36.0 and 60.1 +/- 6.6 ng degraded/h per mg cell protein, respectively. At similar extracellular concentrations of protein or cholesterol, the relative rate of cholesteryl ester hydrolysis from [3H]cholesteryl oleate/cholesteryl [14C]oleate-labeled chylomicron remnants was one-third to one-half that of similarly labeled beta-VLDL. The reduction in the relative rate of chylomicron remnant degradation by macrophages occurred in the absence of chylomicron remnant-induced alterations in low density lipoprotein (LDL) receptor recycling or in retroendocytosis of either 125I-labeled lipoprotein. The rate of internalization of 125I-labeled beta-VLDL by J774 cells was greater than that of 125I-labeled chylomicron remnants, with initial rates of internalization of 0.21 ng/min per mg cell protein for 125I-labeled chylomicron remnants and 0.39 ng/min per mg cell protein for 125I-labeled beta-VLDL. The degradation of 125I-labeled chylomicron remnants and 125I-labeled beta-VLDL was dependent on lysosomal enzyme activity: preincubation of macrophages with the lysosomotropic agent monensin reduced the degradation of both lipoproteins by greater than 90%. However, the pH-dependent rate of degradation of 125I-labeled chylomicron remnants by lysosomal enzymes isolated from J774 cells was 50% that of 125I-labeled beta-VLDL. The difference in degradation rates was dependent on the ratio of lipoprotein to lysosomal protein used and was greatest at ratios greater than 50. The degradation of 125I-labeled beta-VLDL by isolated lysosomes was reduced 30-40% by preincubation of beta-VLDL with 25-50 micrograms oleic acid/ml, suggesting that released free fatty acids could cause the slower degradation of chylomicron remnants. Thus, differences in the rate of uptake and degradation of remnant lipoproteins of different compositions by macrophages are determined by at least two factors: 1) differences in the rates of lipoprotein internalization and 2) differences in the rate of lysosomal degradation.