Two alpha subunit donor splice site mutations cause human trifunctional protein deficiency.

Human trifunctional protein catalyzes three steps in mitochondrial beta-oxidation of fatty acids, including the long chain 3-hydroxyacyl-CoA dehydrogenase step. Deficiency of this heterocomplex, which contains 4 alpha and 4 beta subunits, causes sudden unexplained infant death, a Reye-like syndrome, cardiomyopathy, or skeletal myopathy. We determined the molecular basis of this deficiency in a patient with neonatal presentation and later sudden death using reverse transcription and PCR amplification of his alpha subunit mRNA. We demonstrated a universal deletion of exon 3 (71 bp) in his mRNA. This deletion causes a frameshift and very early premature termination. Amplification of genomic DNA demonstrated that the patient was a compound heterozygote with two different mutations in the 5' donor splice site following exon 3: a paternally inherited G to A transversion at the invariant position +1 and a maternally inherited A to G mutation at position +3. Both allelic mutations apparently cause exon 3 skipping, resulting in undetectable levels of alpha subunit protein, and complete loss of trifunctional protein. This is the initial molecular characterization of trifunctional protein deficiency.

A novel mutation in medium chain acyl-CoA dehydrogenase causes sudden neonatal death.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common known genetic disorder of fatty acid oxidation. Most (approximately 80%) cases are homozygous for a single mutation: A to G replacement at nucleotide 985 (A985G). MCAD deficiency typically presents in the second year of life as hypoketotic hypoglycemia associated with fasting and may progress to liver failure, coma, and death. Prompt diagnosis and management may prevent long-term sequelae. MCAD deficiency was verified by analysis of urinary acylglycine and serum acylcarnitine species from two neonates referred for diagnosis. Full-length cDNA and MCAD exon 7 and 11 genomic clones were prepared for sequence analysis. Normal and mutant cDNAs were expressed in bacteria, and enzymatic activity was assayed by the ferricenium hexaflurophosphate method. Four compound heterozygote individuals from two unrelated families with A985G on one allele and a novel G to A mutation at nucleotide 583 (G583A) as the second mutant allele presented with MCAD deficiency in the first week of life. The expressed G583A mutant protein lacks enzymatic activity. This novel mutation, G583A, is associated with severe MCAD deficiency causing hypoglycemia or sudden, unexpected neonatal death. This previously unrecognized phenotype of MCAD deficiency may contribute significantly to preventable infant deaths.

The human pancreatic lipase-encoding gene: structure and conservation of an Alu sequence in the lipase gene family.

The isolation and characterization of the human gene (hPL) encoding pancreatic lipase is reported. The gene has 13 exons dispersed in about 20 kb of genomic DNA. A pseudogene of hPL was also partially characterized. An Alu sequence is conserved in the homologous introns of hPL and the lipoprotein lipase-encoding gene.

Inherited long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and a fetal-maternal interaction cause maternal liver disease and other pregnancy complications.

Fetal-maternal interactions are critical determinants of maternal health during pregnancy and perinatal outcome. This review explores the causative relationship of a fetal disorder of mitochondrial fatty acid oxidation, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, and the serious maternal liver diseases of pregnancy-preeclampsia, the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet counts), and acute fatty liver of pregnancy. Features of the metabolic adaptation necessitated during the fetal-neonatal transition; common phenotypes of pediatric fatty acid oxidation disorders, including neonatal hypoketotic, hypoglycemia and hepatic crisis; and clinical abnormalities of HELLP and acute fatty liver of pregnancy are presented. Evidence that a common mutation in the alpha-subunit (LCHAD) of trifunctional protein, E474Q, is always one of the mutant alleles in fetal isolated LCHAD deficiency associated with these disorders of pregnancy that cause high maternal, fetal, and newborn morbidity and mortality is reviewed. Recommendations for molecular testing for LCHAD deficiency in families with life-threatening maternal liver disease are given.

The human colipase gene: isolation, chromosomal location, and tissue-specific expression.

The digestion of dietary triglycerides occurs in the duodenum through the action of triglyceride lipase, a pancreatic exocrine protein. The activity of pancreatic lipase is inhibited by the bile salts normally found in the gut lumen. Another pancreatic exocrine protein, colipase, restores the lipolytic activity of triglyceride lipase. The synthesis and secretion of both triglyceride lipase and colipase is increased by dietary fats and secretin. An increase in mRNA accompanies the increased activity, suggesting that the genes for triglyceride lipase and colipase contain nucleotide elements responsive to dietary fats or secretin or both. To study the regulation of colipase expression, we have first isolated the gene for human colipase from a cosmid library with a cDNA probe. The gene was localized to chromosome 6 and is organized into three exons contained in a single 3.3-kb BamHI fragment. The 5'-flanking region of the gene contains a TATA box, a GC box, and a 28-bp region with homology to the rat pancreatic-specific enhancer. This region directs the tissue-specific expression of the chloramphenicol acetyltransferase gene in a transfected rat pancreatic acinar cell line, AR42-J. The same construct is inactive in HEPG2, C2C12, and COS-1 cells. These results demonstrate that the isolated gene for human colipase contains tissue-specific promoter activity in the 5'-flanking DNA. The 28-bp region specifically binds to a factor in nuclear extracts.

Methylation and processing of transfer ribonucleic acid in mammalian and bacterial cells.

The relationship between the methylation and processing of tRNA in both bacterial and mammalian cell systems was investigated by assessing the methylation of an existing population of precursor-tRNAs in the absence of tRNA synthesis. When the synthesis of tRNA in Escherichia coli B (rifampicin) and human KB cells (actinomycin D) was inhibited with the appropriate antibiotic, the incorporation of [3H[methyl groups into tRNA (via [methyl-3H]methionine labeling) rapidly declined with time and was essentially complete within 30 and 60 min, respectively. Although antibiotic treatment predictably reduced the incorporation of methyl groups into tRNA, it also resulted in significant changes in the distribution of the type of methylated products formed. Thus, for KB cells the marked increases in the per cent of radioactivity incorporated into 2'-0-methylribose derivatives, N2-methylguanine, and 3-methylcytosine of tRNA preparation pre-chased with actinomycin D for progressively longer periods of time prior to labeling with [methyl-3H]methionine led to the interpretation that these methylated constituents were formed predominantly during the late stages of tRNA maturation. Similarly, progessive and marked decreases in 1-, 7-, and N2, N2-methylguanine, and moderate decreases in 1-methyladenosine, 5-methylcytosine, and 5-methyluracil revealed that these methylated products were formed primarily during the early and intermediate stages of maturation, respectively. Similar analysis of E. coli B methylation products indicated that the bulk of methyl groups incorporated into the base moieties of tRNA (1- and 7-methylguanine, 2- and N6-methyladenine, and 5-methyluracil) occurred prior to the formation of 2'-0-methylribose derivatives. Additional evidence is presented which negates the possibility that an ancillary action of these antibiotics was the inhibition of specific tRNA-methyl-transferase enzymes.

Separation of the methylated nucleoside consituents of ribonucleic acid by two-dimensional thin-layer chromatography.

In an attempt to separate a number of methylated nucleoside constituents reportedly present in various mammalian RNA preparations, a comparison of the migration of selected nucleoside standards was assessed via thin-layer chromatographic techniques with respect to three solvent systems and an adsorbent of varying proportions of silica gel and microcrystalline cellulose. The results of these studies revealed that a mixture of seventeen methylated constituents could be separated following two-dimensional development on an adsorbent containing 60% cellulose and 40% silica gel. Constituents separated included the 2'-O-methylated derivatives of adenosine, guanosine, cytidine and uridine, 1-, N6- and N6,N6-methyladenosines, 1-methylinosine, 1-, 7-, N2- and N2,N2-methylguanosines, 3-, 4- and 5-methylcytidines and 3- and 5-methyluridines. In addition, all of the above constituents were separated from their respective non-methylated nucleosides and bases.

Immunospecific retention of oligonucleotides possessing N6-methyladenosine and 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were immobilized on Sepharose and the resulting immunoadsorbents tested for their ability to retain specific oligonucleotides possessing the corresponding antigenic haptens (i.e. m6A and m7G). Results obtained with oligonucleotides derived from ribonuclease T1 digests of Escherichia coli tRNA (previously labeled with [methyl-3H]methionine) indicated that each immunoadsorbent quantitatively and exclusively retained those methyl-3H-labeled oligonucleotides possessing [methyl-3H]m6A and [methyl-3H]m7G. Elution and subsequent characterization of the retained methyl-3H-labeled oligonucleotides via DEAE-cellulose chromatography revealed the presence of several small oligonucleotides containing m7G and a single, larger oligonucleotide containing m6A. These findings are in accord with previously sequenced structures which indicate that numerous bacterial tRNA species possess m7G while only tRNAVal contains m6A.

Characterization of antibodies specific for N6-methyladenosine and for 7-methylguanosine.

Antibodies specific for N6-methyladenosine (m6A) and for 7-methylguanosine (m7G) were prepared by immunization of rabbits with nucleoside conjugates of bovine serum albumin (i.e, m6A--BSA and m7G-BSA). Specificity of each antibody was assessed by inhibition of the homologous precipitin reaction with various nucleosides. These analyses revealed that the antibodies elicited in response to m6A--BSA were specific for the N6-methyl moiety of adenosine with minimal or no cross-reactivity with BSA, adenosine, and guanosine. Although a major fraction of antibodies elicited in response to m7G--BSA were specific for m7G, considerable cross-reactivity was observed with BSA. These latter antibodies were removed by affinity chromatography utilizing BSA-Sepharose adsorbent. In similar fashion, antibodies specific for m6A and m7G were isolated by immunospecific adsorption to antigen-coupled Sepharose (e.g. m6A--BSA-Sepharose), eluted, and coupled to Sepharose. The ability of these antibody-coupled adsorbents to retain specific methylated [methyl-3H]nucleosides derived from [methyl-3H]tRNA digests was assessed. Both the anti-m7G and anti-m6A antibody adsorbents quantitatively and exclusively retained 7-[3H]methylguanosine and N6-[3H]methyladenosine, respectively. The application of these adsorbents to fractionate oligonucleotides and nucleic acids is discussed.

Effects of estradiol on uterine ribonucleic acid metabolism. Assessment of transfer ribonucleic acid methylation.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.

Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart.

We have isolated and sequenced a cDNA encoding rat cardiac troponin I. The predicted amino acid sequence was highly identical with previously reported chemically derived amino acid sequences for rabbit and bovine cardiac troponin I. Clones for slow skeletal muscle troponin I were also obtained from neonatal rat cardiac ventricle by the polymerase chain reaction. The nucleotide sequences of these clones were determined to be more than 99% identical with a previously reported rat slow skeletal troponin I cDNA [Koppe et al. (1989) J. Biol. Chem. 264, 14327-14333]. The troponin I clones hybridized to RNA from the appropriate muscle from adult animals. However, RNA from fetal and neonatal rat heart also hybridized with the slow skeletal troponin I cDNA, demonstrating its expression in fetal and neonatal rat heart. Slow skeletal troponin I steady-state mRNA levels decreased with increasing age, but cardiac troponin I mRNA levels increased through fetal and early neonatal cardiac development. Thus, during fetal and neonatal development, slow skeletal and cardiac troponin I isoforms are coexpressed in the rat heart and regulated in opposite directions. The degree of primary sequence differences in these isoforms, especially at phosphorylation sites, may result in important functional differences in the neonatal myocardium.

Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development.

We report the cDNA sequences of rat colipase, rat pancreatic lipase (rPL), and a rat pancreatic lipase-related protein (rPLRP). Comparison to the human PLRP cDNA suggests that the isolated clone encodes rPLRP-2. Both cDNA and a third cDNA encoding rPLRP-1 are secreted from Sf9 cells infected with recombinant baculovirus. rPL and rPLRP-2 hydrolyze triolein, 8.0 and 4.4 mumol.min-1.microgram-1, respectively. They are inhibited by bile salts, and activity is restored by (pro)colipase. PLRP-1 has barely detectable activity against triolein, even with (pro)colipase present. The pattern of mRNA expression during development in the rat reveals that all mRNA are low in the fetal rat pancreas. Both PLRP mRNA rise just before birth to a maximum 12 h after birth. They fall to low levels in the adult. In contrast, the PL mRNA is low at birth and rises rapidly during the suckling-weanling transition. In conclusion, the rat has at least three genes encoding different lipases, and these related genes have separate regulatory controls.

Antibody nucleic acid complexes. Immunospecific retention of N6-methyladenosine-containing transfer ribonucleic acid.

Antibodies specific for N6-methyladenosine (m6A) were immobilized on Sepharose and the resulting immunoadsorbent was tested for its ability to retain those Escherichia coli tRNAs containing the antigenic hapten, i.e., m6A. Results obtained with [32P]PO4- and [methyl-3H]-methionine-labeled tRNAs indicated that approximately 3 to 5% of the radioactive RNA was retained by the immunoadsorbent. Under identical conditions, but in the presence of m6A (1 mg/mL), less than 0.2% of the radioactivity was retained. Subsequent characterization of the retained tRNA via (a) analysis of methyl-3H-labeled, methylated nucleosides, (b) two-dimensional gel electrophoresis, and (c) analysis of the retention of [3H]aminoacyl-tRNA species led to the conclusion that the anti-m6A/Sepharose adsorbent quantitatively and exclusively retained a single tRNA species containing m6A, namely, tRNAVal.

Extracellular processing of proapolipoprotein A-II in Hep G2 cell cultures is mediated by a 54-kDa protease immunologically related to cathepsin B.

Apolipoprotein A-II is the second most abundant polypeptide found in human plasma high density lipoprotein particles. The primary translation product of human apo-A-II mRNA is a prepropolypeptide. We have previously reported (Gordon, J. I., Sims, H. F., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1984) J. Biol. Chem. 259, 15556-15563) that the prosegment of apo-A-II was removed following export from a human hepatoma cell line (Hep G2). This represented a novel processing compartment for prosegments terminating with paired basic residues and differed from the processing of proalbumin which occurred with high efficiency prior to export from these cells. We have now characterized the enzyme responsible for this extracellular cleavage. The proapo-A-II converting activity is blocked by the thiol protease inhibitors antipain, E-64, leupeptin, and Ala-Lys-Arg chloromethyl ketone. Incubation of 125I-iodotyrosylated Ala-Lys-Arg chloromethyl ketone with serum-free media harvested from cell cultures over a 12-h period revealed a time-dependent accumulation of a 54-kDa protease. Although small quantities of the 54-kDa protease were detected in cell lysates, the major intracellular sequences labeled by the affinity probe had masses of 31.5 and 6 kDa. The 54-kDa extracellular, as well as 31.5- and 6-kDa intracellular, species were all immunoprecipitated by monospecific anti-human liver cathepsin B IgG. Addition of this antibody to media inhibited extracellular conversion of proapo-A-II to the mature protein. Based on these observations, we conclude that a "pro" cathepsin B-like protease exported by Hep G2 cells is responsible for proapo-A-II prosegment removal. It appears that cathepsin B-like proteases exhibit a complex pattern of segregation within the secretory pathway and that larger molecular weight forms of cathepsin B-like proteases are capable of accurately processing propolypeptides.

Human proapolipoprotein A-II is cleaved following secretion from Hep G2 cells by a thiol protease.

The two principal high-density lipoprotein apolipoproteins A-I and A-II are both initially synthesized as preproproteins. The prosegment of apo-A-I is unusual: it ends with paired glutamine residues and is removed extracellularly. The apo-A-II prosegment resembles the propeptides of prohormones and proalbumin: it ends with paired basic amino acids. We have studied the processing of proapo-A-II in a human hepatoma cell line (Hep G2) which is known to accurately and efficiently remove the prosegment from proalbumin prior to secretion. Pulse-chase experiments were performed in order to determine if the apo-A-II prosegment is removed prior to or after secretion. Apo-A-II was purified from cell lysates and media at various times during the chase and subjected to automated sequential Edman degradation. The results indicate that proteolytic processing of proapo-A-II is largely an extracellular event. These cells secrete the protease responsible for prosegment removal. The converting activity present in media is not blocked by serine protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, and furoyl saccharin) or by a metalloprotease inhibitor (o-phenanthroline). It is inhibited by the thiol protease reagents p-chloromercuribenezene-sulfonic acid and leupeptin. Prosegment removal changes the pI of the dominant apo-A-II isoform from 6.61 to 4.95. The presence of the propeptide does not prevent specific in vitro recombination of apo-A-II with high-density lipoprotein3 particles present in normolipemic serum. Extracellular processing after a single basic amino acid has been described for a variety of precursor proteins. Extracellular cleavage of the apo-A-II propeptide after paired COOH-terminal basic residues represents a novel processing pathway.

Proteolytic processing and compartmentalization of the primary translation products of mammalian apolipoprotein mRNAs.

The steps involved in the initial assembly of apolipoproteins and lipids into supramolecular arrays (nascent lipoprotein particles) are largely unknown. Examination of the proteolytic processing and compartmentalization of the primary translation products of apolipoprotein mRNAs represents one approach to deciphering the molecular details of lipoprotein assembly. The structures of the primary translation products of seven mammalian apolipoprotein mRNAs has been determined in the past several years. The organization of apolipoprotein signal peptides is typical of eukaryotic prepeptides, although an unusual degree of sequence conservation is present among the signal segments of apo AI, AIV, and E. For those apolipoprotein sequences studied in detail, SRP-dependent cotranslational translocation and proteolytic processing appears to be highly efficient and results in sequestration of the processed protein within the lumen of the endoplasmic reticulum (ER). However the mechanism by which these lipid-binding proteins avoid arrest during their translocation through the lipid bilayer of the ER membrane remains obscure. The two principal human HDL apolipoproteins undergo novel extracellular post-translational proteolytic processing, which results in removal of nonhomologous propeptides. The proteases responsible for proapo AI and AII processing appear to be different. The processing of these proapolipoproteins provides a potential series of steps for regulating the ordered assembly of HDL constituents.

Isolation and sequencing of a complementary deoxyribonucleic acid clone encoding human placental 17 beta-estradiol dehydrogenase: identification of the putative cofactor binding site.

17 beta-Estradiol dehydrogenase (EC 1.1.1.62) catalyzes the interconversion of estradiol and estrone in human term placenta. We have raised a specific polyclonal antibody to this abundant placental enzyme to study its role in late pregnancy events and its molecular biologic characteristics. In this work the 17 beta-estradiol dehydrogenase antibody was used to isolate and sequence a complementary deoxyribonucleic acid clone encoding about 98% of the amino acid sequence of the 17 beta-estradiol dehydrogenase molecule. This sequence verifies previous sequence data on the molecule's steroid binding site and also localizes a putative nicotinamide adenine dinucleotide binding region similar to that of many other pyridine nucleotide-dependent dehydrogenases. Isolation of the complementary deoxyribonucleic acid for 17 beta-estradiol dehydrogenase expands our knowledge of the structure-function relationships of the enzyme and is a major step in our understanding of its biologic function in pregnancy.

A fetal fatty-acid oxidation disorder as a cause of liver disease in pregnant women.

BACKGROUND: Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count) are serious hepatic disorders that may occur during pregnancy in women whose fetuses are later found to have a deficiency of long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase. This enzyme resides in the mitochondrial trifunctional protein, which also contains the active site of long-chain 2,3-enoyl-CoA hydratase and long-chain 3-ketoacyl-CoA thiolase. We undertook this study to determine the relation between mutations in the trifunctional protein in infants with defects in fatty-acid oxidation and acute liver disease during pregnancy in their mothers. METHODS: In 24 children with 3-hydroxyacyl-CoA dehydrogenase deficiency, we used DNA amplification and nucleotide-sequence analyses to identify mutations in the alpha subunit of the trifunctional protein. We then correlated the results with the presence of liver disease during pregnancy in the mothers. RESULTS: Nineteen children had a deficiency only of long-chain 3-hydroxyacyl-CoA dehydrogenase and presented with hypoketotic hypoglycemia and fatty liver. In eight children, we identified a homozygous mutation in which glutamic acid at residue 474 was changed to glutamine. Eleven other children were compound heterozygotes, with this mutation in one allele of the alpha-subunit gene and a different mutation in the other allele. While carrying fetuses with the Glu474Gln mutation, 79 percent of the heterozygous mothers had fatty liver of pregnancy or the HELLP syndrome. Five other children, who presented with neonatal dilated cardiomyopathy or progressive neuromyopathy, had complete deficiency of the trifunctional protein (loss of activity of all three enzymes). None had the Glu474Gln mutation, and none of their mothers had liver disease during pregnancy. CONCLUSIONS: Women with acute liver disease during pregnancy may have a Glu474Gln mutation in long-chain hydroxyacyl-CoA dehydrogenase. Their infants are at risk for hypoketotic hypoglycemia and fatty liver.

Molecular heterogeneity in very-long-chain acyl-CoA dehydrogenase deficiency causing pediatric cardiomyopathy and sudden death.

BACKGROUND: Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the beta-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. METHODS AND RESULTS: We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent or severe reduction in enzyme activity in all. CONCLUSIONS: Infantile CM is the most common clinical phenotype of VLCAD deficiency. Mutations in the human VLCAD gene are heterogeneous. Although mortality at presentation is high, both the metabolic disorder and cardiomyopathy are reversible.

In vitro conversion of proapoprotein A-I to apoprotein A-I. Partial characterization of an extracellular enzyme activity.

Previous studies have established that human hepatocellular carcinoma cells (Hep G2) secrete into serum-free medium the pro form of apolipoprotein A-I (proapo-A-I) suggesting that its conversion to mature apo-A-I occurs after secretion. In order to assess the mode and site of proapo-A-I to apo-A-I conversion, we incubated the medium from [3H]proline-labeled Hep G2 cells with either human plasma, serum, lymph, or fractions thereof obtained by density gradient ultracentrifugation. The conversion was monitored by two-dimensional gel electrophoresis and by Edman degradation. Human plasma, serum, or mesenteric lymph all induced proapo-A-I to apo-A-I conversion; this was time dependent, unaffected by the serine protease inhibitor phenylmethylsulfonyl fluoride and inhibited by EDTA. Purified radiolabeled proapo-A-I bound to lymph chylomicrons and plasma high density lipoproteins. The converting enzyme was associated with both of these particles. Activity was also found in the d greater than 1.21-g/ml fraction and may have been derived from high density lipoprotein after displacement by high salts and/or ultracentrifugal force. We conclude that the conversion of proapo-A-I to apo-A-I occurs extracellularly and is probably effected by a metallo-enzyme which may act at the amphiphilic surface of either chylomicrons or high density lipoproteins.