Modified helix-loop-helix motifs of calmodulin--The influence of the exchange of helical regions on calcium-binding affinity.

The four calcium-binding sites, called the helix-loop-helix, or the EF-hand motifs, of calmodulin differ in their ion-binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium-binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium-binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocyanine bromide). Binding abilities were seen to increase several-fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111-414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it.

Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.

In vivo metabolism of apolipoprotein A-IV in severe hypertriglyceridemia: a combined radiotracer and stable isotope kinetic study.

Apolipoprotein (apo) A-IV is an intestinally derived apolipoprotein that plays a potentially important role in lipoprotein metabolism and reverse cholesterol transport. However, the factors that regulate its plasma concentrations are not well understood. Plasma apoA-IV levels have been previously shown to correlate with fasting triglyceride (TG) levels in humans with TG levels less than 300 mg/dl (Lagrost et al. 1989. J. Lipid Res. 30: 701-710). In this study, we established that apoA-IV levels were significantly elevated (mean 29.3 mg/dl) in a group of 15 hypertriglyceridemic patients (TG > 300 mg/dl) compared with normolipidemic controls (mean 13.4 mg/dl). In order to investigate the relationship between hypertriglyceridemia and apoA-IV metabolism, we then studied the in vivo kinetics of apoA-IV in two healthy hypertriglyceridemic patients (mean TG = 1297 mg/dl) compared with normolipidemic control subjects. Combined studies using endogenous stable isotope labeling (with a primed constant infusion of deuterated L-leucine) and exogenous radiolabeling (with 125I) of apoA-IV were performed. Both stable isotope and radiotracer studies demonstrated substantially decreased apoA-IV fractional catabolic rates (FCR) in the hypertriglyceridemic patients (1.24 +/- 0.13 day-1) compared with controls (2.33 +/- 0.08 day-1). The apoA-IV production rate was not significantly different between the two groups. Gel filtration chromatography of plasma indicated an increased proportion of apoA-IV in the triglyceride-rich lipoproteins (TRL) of the hypertriglyceridemic patients compared with controls and delayed catabolism of this TRL-associated apoA-IV. The rate of apoA-IV catabolism from the lipid deficient fraction was not different between the hypertriglyceridemic patients and controls. In summary, plasma levels of apoA-IV are significantly elevated in hypertriglyceridemic patients due to delayed catabolism of apoA-IV as demonstrated by both endogenous stable isotope labeling and exogenous radiotracer techniques.

Markedly accelerated catabolism of apolipoprotein A-II (ApoA-II) and high density lipoproteins containing ApoA-II in classic lecithin: cholesterol acyltransferase deficiency and fish-eye disease.

Classic (complete) lecithin:cholesterol acyltransferase (LCAT) deficiency and Fish-eye disease (partial LCAT deficiency) are genetic syndromes associated with markedly decreased plasma levels of high density lipoprotein (HDL) cholesterol but not with an increased risk of atherosclerotic cardiovascular disease. We investigated the metabolism of the HDL apolipoproteins (apo) apoA-I and apoA-II in a total of five patients with LCAT deficiency, one with classic LCAT deficiency and four with Fish-eye disease. Plasma levels of apoA-II were decreased to a proportionately greater extent (23% of normal) than apoA-I (30% of normal). In addition, plasma concentrations of HDL particles containing both apoA-I and apoA-II (LpA-I:A-II) were much lower (18% of normal) than those of particles containing only apoA-I (LpA-I) (51% of normal). The metabolic basis for the low levels of apoA-II and LpA-I:A-II was investigated in all five patients using both exogenous radiotracer and endogenous stable isotope labeling techniques. The mean plasma residence time of apoA-I was decreased at 2.08 +/- 0.27 d (controls 4.74 +/- 0.65 days); however, the residence time of apoA-II was even shorter at 1.66 +/- 0.24 d (controls 5.25 +/- 0.61 d). In addition, the catabolism of apoA-I in LpA-I:A-II was substantially faster than that of apoA-I in LpA-I. In summary, genetic syndromes of either complete or partial LCAT deficiency result in low levels of HDL through preferential hypercatabolism of apoA-II and HDL particles containing apoA-II. Because LpA-I has been proposed to be more protective than LpA-I:A-II against atherosclerosis, this selective effect on the metabolism of LpA-I:A-II may provide a potential explanation why patients with classic LCAT deficiency and Fish-eye disease are not at increased risk for premature atherosclerosis despite markedly decreased levels of HDL cholesterol and apoA-I.

Evaluation of apoA-I kinetics in humans using simultaneous endogenous stable isotope and exogenous radiotracer methods.

Apolipoprotein A-I is the major apolipoprotein constituent of high density lipoproteins (HDL). Methods used to investigate in vivo kinetics of apoA-I include exogenous labeling with radioiodine and endogenous labeling with stable isotopically labeled amino acids. We report here a direct comparison of these methods to determine the in vivo kinetics of apoA-I in four normal subjects. Purified apoA-I was labeled with 125I, reassociated with autologous plasma, and injected into study subjects. At the same time, [13C6]phenylalanine was administered as a primed constant infusion for up to 14 hours. The kinetic parameters of apoA-I were determined from the 125I-labeled apoA-I plasma curves. For the analysis of data from stable isotope studies, very low density lipoprotein (VLDL) apoB-100, VLDL apoB-48, and total apoA-I were isolated by ultracentrifugation and subsequent preparative NaDodSO4-PAGE, hydrolyzed, and derivatized. The tracer/tracee ratio was determined by gas chromatography-mass spectrometry. Monoexponential function analysis was used to determine the tracer/tracee curves of VLDL apoB-100 and VLDL apoB-48, and total apoA-I. The mean plateau tracer/tracee ratio of VLDL apoB-100 (primarily liver-derived) was 5.19%, whereas that of VLDL apoB-48 (intestinally derived) was only 3.74%. Using the VLDL apoB-100 plateau tracer/tracee ratio as the estimate of the precursor pool enrichment for apoA-I, the mean apoA-I residence time (RT) was 5.14 +/- 0.41 days, compared with 4.80 +/- 0.30 days for the exogenous labeling method. The apoA-I RTs using these two methods were highly correlated (r = 0.874).(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed catabolism of high density lipoprotein apolipoproteins A-I and A-II in human cholesteryl ester transfer protein deficiency.

Deficiency of the cholesteryl ester transfer protein (CETP) in humans is characterized by markedly elevated plasma concentrations of HDL cholesterol and apoA-I. To assess the metabolism of HDL apolipoproteins in CETP deficiency, in vivo apolipoprotein kinetic studies were performed using endogenous and exogenous labeling techniques in two unrelated homozygotes with CETP deficiency, one heterozygote, and four control subjects. All study subjects were administered 13C6-labeled phenylalanine by primed constant infusion for up to 16 h. The fractional synthetic rates (FSRs) of apoA-I in two homozygotes with CETP deficiency (0.135, 0.134/d) were found to be significantly lower than those in controls (0.196 +/- 0.041/d, P < 0.01). Delayed apoA-I catabolism was confirmed by an exogenous radiotracer study in one CETP-deficient homozygote, in whom the fractional catabolic rate of 125I-apoA-I was 0.139/d (normal 0.216 +/- 0.018/d). The FSRs of apoA-II were also significantly lower in the homozygous CETP-deficient subjects (0.104, 0.112/d) than in the controls (0.170 +/- 0.023/d, P < 0.01). The production rates of apoA-I and apoA-II were normal in both homozygous CETP-deficient subjects. The turnover of apoA-I and apoA-II was substantially slower in both HDL2 and HDL3 in the CETP-deficient homozygotes than in controls. The kinetics of apoA-I and apoA-II in the CETP-deficient heterozygote were not different from those in controls. These data establish that homozygous CETP deficiency causes markedly delayed catabolism of apoA-I and apoA-II without affecting the production rates of these apolipoproteins.

Studies on the interaction of the dye, stains-all, with individual calcium-binding domains of calmodulin.

We show that the calcium-mimic dye, Stains-all, is a convenient probe to study the structural features of the individual calcium-binding sites of calmodulin (CaM) and related calcium-binding proteins (CaBP). These peptides bind the dye in their calcium-binding sites, and induce a circular dichroism (CD) band in the bound dye in the 620 nm (J band) region, which is abolished upon the addition of calcium. Replacement of Asp by Asn in the + x position of the weaker calcium-binding site (site I of CaM) abolishes the dye binding, while the same change in the higher affinity site IV attenuates the binding of the dye and does not abolish it. Replacement of Tyr in site IV with Trp does not distort the geometry, although it increases the dye binding affinity.

In vivo metabolism of apolipoprotein A-I in a patient with homozygous familial hypercholesterolemia.

Familial hypercholesterolemia (FH), caused by a defect in the low density lipoprotein (LDL) receptor, results in high plasma concentrations of LDL cholesterol due to both overproduction and delayed catabolism of LDL. FH is also associated with significantly lower levels of plasma high density lipoprotein cholesterol and apolipoprotein (apo) A-I in both heterozygous and homozygous patients. However, the metabolic basis of the hypoalphalipoproteinemia in FH has not been elucidated. We investigated the kinetics of apo A-I in a homozygous FH patient and two normal control subjects by using endogenous labeling with a stable isotopically labeled amino acid. Study subjects were administered a primed constant infusion of 13C6-phenylalanine for 12 hours. Apolipoproteins were isolated from plasma drawn at selected time points and analyzed for their isotopic enrichment by gas chromatography-mass spectrometry. The fractional catabolic rate of apo A-I in the FH subject was found to be substantially increased (0.38 day-1) compared with that of the normal subjects (mean, 0.26 day-1). In addition, the apo A-I production rate was decreased in the FH subject (6.5 mg/kg.day-1) compared with the normal subjects (mean, 11.1 mg/kg.day-1). In conclusion, the low levels of high density lipoprotein cholesterol and apo A-I in this homozygous FH patient are due to the combined metabolic defects of increased apo A-I catabolism and decreased apo A-I production.

Heptad motifs within the distal subdomain of the coiled-coil rod region of M protein from rheumatic fever and nephritis associated serotypes of group A streptococci are distinct from each other: nucleotide sequence of the M57 gene and relation of the deduced amino acid sequence to other M proteins.

Streptococcal M protein, a dimeric alpha helical coiled-coil molecule, is an antigenically variable virulence factor on the surface of the bacteria. Our recent conformational analysis of the complete sequence of the M6 protein led us to propose a basic model for the M protein consisting of an extended central coiled-coil rod domain flanked by a variable N-terminal and a conserved C-terminal end domains. The central coiled-coil rod domain of M protein, which constitutes the major part of the M molecule, is made up of repeating heptads of the generalized sequence a-b-c-d-e-f-g, wherein "a" and "d" are predominantly apolar residues. Based on the differences in the heptad pattern of apolar residues and internal sequence homology, the central coiled-coil rod domain of M protein could be further divided into three subdomains I, II, and III. The streptococcal sequelae rheumatic fever (RF) and acute glomerulonephritis (AGN) have been known to be associated with distinct serotypes. Consistent with this, we observed that the AGN associated M49 protein exhibits a heptad motif that is distinct from the RF associated M5 and M6 proteins. Asn and Leu predominated in the "a" and "d" positions, respectively, in subdomain I of the M5 and M6 proteins, whereas apolar residues predominated in both these positions in the M49 protein. To establish whether the heptad motif of M49 is unique to this protein, or is a general characteristic of nephritis-associated serotypes, the amino acid sequence of M57, another nephritis-associated serotype, has now been examined. The gene encoding M57 was amplified by PCR, cloned into pUC19 vector, and sequenced. The C-terminal half of M57 is highly homologous to other M proteins (conserved region). In contrast, its N-terminal half (variable region) revealed no significant homology with any of the M proteins. Heptad periodicity analysis of the M57 sequence revealed that the basic design principles, consisting of distinct domains observed in the M6 protein, are also conserved in the M57 molecule. However, the heptad motif within the coiled-coil subdomain I of M57 was distinct from M5 and M6 but similar to M49. Similar analyses of the heptad characteristics within the reported sequences of M1, M12, and M24 proteins further confirmed the conservation of the overall architectural design of sequentially distinct M proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

The amino-terminal region of group A streptococcal M protein determines its molecular state of assembly and function.

Group A streptococcal M protein, a major virulence factor, is an alpha-helical coiled-coil dimer on the surface of the bacteria. Limited proteolysis of type 57 streptococcus with pepsin released two fragments of the M57 molecule, with apparent molecular weights of 32,000 and 27,000 on SDS-PAGE. However, on gel filtration under nondenaturing conditions, each of these proteins eluted as two distinct molecular forms. The two forms corresponded to their dimeric and monomeric state as compared to the gel filtration characteristics of known dimeric coiled-coil proteins. The results of sedimentation equilibrium measurements were consistent with this, but further indicated that the "dimeric form" consisted of a dimer in rapid equilibrium with its monomer, whereas the "monomeric form" does not dimerize. The monomeric form was the predominant species for the 27 kD species, whereas the dimeric form predominated for the 32 kD species. Sequence analysis revealed the 27 kD species to be a truncated derivative of the 32 kD PepM57 species, lacking the N-terminal nonheptad region of the M57 molecule. These data strongly suggested that the N-terminal nonheptad region of PepM57 is important in determining the molecular state of the molecule. Consistent with this, PepM49, another nephritis-associated serotype, which lacks the nonheptad N-terminal region, also eluted as a monomer on gel filtration under nondenaturing conditions. Furthermore, removal of the N-terminal nonheptad segment of the dimeric PepM6 protein converted it into a monomeric form. The dimeric molecular form of both the 32 kD PepM57 and the 27 kD PepM57 did not represent a stable state of assembly, and were susceptible to conversion to the corresponding monomeric molecular forms by simple treatments, such as lyophilization. The 27 kD PepM57 exhibited a greater propensity than the 32 kD species to exist in the monomeric form. The 32 kD species contained the opsonic epitope of the M57 molecule, whereas the 27 kD species lacked the same. This is consistent with the previous reports on the importance of the N-terminal region of M protein for its opsonic activity. Together, these results strongly suggest that, in addition to its importance for the biological function, the N-terminal region of the M protein plays a dominant role in determining the molecular state of the M molecule, as well as its stability.

Domain structure and molecular flexibility of streptococcal M protein in situ probed by limited proteolysis.

Serologically distinct group A streptococcal M proteins, the antiphagocytic determinants of the bacteria, have a highly repetitive sequence and exhibit a heptad periodicity characteristic of alpha-helical coiled-coil proteins. Based on the differences in the pattern of hepatad periodicity, the coiled-coil region of the complete M molecule has been divided into three distinct domains: I, II, and III. Domains I and II together constitute the variable part of M protein, whereas domain III is conserved among serotypes. Pepsin treatment of the M5, M6, and M24 streptococci results in a preferential cleavage of their M molecules between the predicted domains II and III, releasing biologically active fragments of the respective M proteins. Thus, a pepsin cleavage site at the junction of their variable and conserved regions is conserved in the M5, M6, and M24 proteins. In contrast, in the case of the M49 streptococci, the primary site of pepsin cleavage was observed to be within the conserved region of the M49 molecule, rather than at the junction of its variable and conserved regions. Despite containing part of the conserved region, the PepM49 protein is significantly smaller than the pepsin fragments of the M5, M6, and M24 proteins, which contain only the variable regions. However, in addition to the major PepM49 species, the pepsin digest of the type-49 streptococci also contained a smaller fragment, PepM49/a, as a minor component. Its formation was extremely sensitive to the pH of pepsin digestion. PepM49/a, which retains both the propensity to attain an alpha-helical conformation and the opsonic antibody epitope of the M49 molecule, contains only domains I and II like the other PepM proteins. Thus, as in the M5, M6, and M24 proteins, a pepsin cleavage site at the junction of the variable and conserved regions is indeed present in the M49 molecule, but is much less accessible relative to the other serotypes. Thus, the pepsin cleavage sites in the M protein correlate quite well with the boundaries of structurally distinct domains reflected by the predictive analysis. These sites apparently represent the flexible/hinge regions of the molecule. PepM49/a is the least repetitive and the shortest of the M protein pepsin fragments isolated so far. These results suggest that the flexibility of the interdomain regions in M protein may be dependent on the molecular size of their variable domains.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of ions on cyclization of the amino terminal glutamine residues of tryptic peptides of streptococcal PepM49 protein. Resolution of cyclized peptides by HPLC and characterization by mass spectrometry.

RPHPLC of the tryptic digest of lysine blocked group A streptococcal PepM49 protein (DHP-PepM49) consistently yielded, among others, two pairs of peptides which were well resolved, eluted in tandem, and had identical amino acid compositions. In each pair, the earlier eluting peptide was readily amenable to sequencing and yielded an amino-terminal glutamine whereas the later eluting peptide could not be sequenced. Mass spectral analysis revealed that each of these pairs of peptides differed in mass corresponding to the loss of ammonia. These data suggested that the later eluting peptide in each pair is a result of cyclization of the amino-terminal glutamine residue to pyroglutamic acid, which apparently leads to an increase in the hydrophobicity of the peptide. A kinetic analysis of the tryptic digestion of the DHP-PepM49 protein revealed that the cyclized form of the peptides were essentially absent during the initial time and increased with time of incubation, with a concomitant decrease in the uncyclized form. In 0.2 M ammonium bicarbonate at 37 degrees, nearly 44% conversion of the glutaminyl peptides to the pyroglutamyl peptides was observed in 24 h. This conversion was accelerated in sodium phosphate buffer relative to that in ammonium bicarbonate whereas it had a significantly lower rate in ammonium acetate buffer. The conversion was also temperature dependent, with essentially no cyclization at 0 degree, in all the three buffers. Thus, an extended digestion at 0 degree or a brief digestion at 37 degrees in ammonium acetate was found to be a suitable condition for limiting the cyclization of amino-terminal glutamine residues of PepM49 peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Human apolipoprotein A-I. Post-translational modification by covalent phosphorylation.

In vitro phosphorylation of purified human plasma apolipoprotein A-I (apoA-I) by a recently characterized Ca2+/calmodulin-dependent kinase (Beg, Z. H., Stonik, J. A., and Brewer, H. B., Jr. (1987) J. Biol. Chem. 262, 13228-13240) was time-, Ca2+-, and calmodulin-dependent. Maximal phosphorylation of human apoA-I revealed a stoichiometry of approximately 1 mol of PO4/mol of apoA-I. Phosphorylation of apoA-I resulted in an increase of two negative charges and consequently a shift to a more acidic pI for each apoA-I isoform following isoelectrofocusing. Dephosphorylation of 32P-apoA-I with either phosphatase I or a Ca2+/calmodulin-dependent phosphatase was associated with a virtually complete loss of 1 mol of 32PO4/mol of apoA-I. Phosphoamino acid analysis of a purified 32P-peptide established that the phosphorylation occurred on a single serine residue. Automated Edman degradation of the purified 32P-peptide revealed a single amino acid sequence and indicated that phosphorylation occurred on the serine at residue 201 in the apoA-I sequence. ApoA-I was shown to be secreted as a phosphoapolipoprotein by HepG-2 cells as well as primary human hepatocytes. Analysis of HepG-2 cells established that intracellular apoA-I, like secreted apoA-I, is phosphorylated. Dephosphorylation of both secreted and intracellular 32P-apoA-I revealed the loss of radioactivity in the apoA-I protein bands. These data provide the initial description of a post-translational modification involving reversible phosphorylation of extracellular as well as intracellular apoA-I on a serine residue. These combined results suggest that synthesis and secretion of apoA-I as a phosphoapolipoprotein in HepG-2 cells as well as primary human hepatocytes may play an important role in lipoprotein assembly, intracellular transport as well as processing, and lipoprotein secretion.

Metabolism of murine and human embryos: search for a growth indicator in vitro culture medium.

Ham's F-10 medium was analyzed biochemically before and after growth of murine and human embryos. Ham's F-10 medium (280 mosm/kg, pH 7.4) alone, by means of reverse-phase high-performance liquid chromatography, demonstrated one major hydrophilic peak, which eluted at 4 to 8 minutes in a 10% to 48% acetonitrile gradient. This peak showed a single peptide of 50 kilodaltons in one-dimensional polyacrylamide gel electrophoresis and size exclusion high-performance liquid chromatography. After the growth of two-cell murine embryos to eight-cell embryos or blastocysts, the major hydrophilic peak was greatly reduced or absent in the culture medium, and in turn a major hydrophobic peak appeared that eluted at 29 minutes. The major hydrophobic peak could not be focused in one-dimensional polyacrylamide gel electrophoresis, but a high content of polar and nonpolar amino acids was revealed in N-terminal sequencing and mass spectrometric analysis. This shift in the peaks was not detected when embryos were cultured in the presence of 0.02% sodium azide. In vitro culture of human zygotes from the pronuclear stage to two to eight cells caused a similar disappearance of the major hydrophilic peak concomitant with the appearance of one to three major hydrophobic peaks in the culture medium. We conclude that the change in profile of culture medium from hydrophilic to hydrophobic peaks on high-performance liquid chromatography is indicative of the metabolic pattern of murine and human embryos. These data also indicate that murine and human embryos do not secrete any major peptide during their development in vitro.

Apolipoprotein B synthesized by Hep G2 cells undergoes fatty acid acylation.

Apolipoprotein B is the principal protein associated with cholesterol transport in the blood and has been proposed to play a central role in human atherogenesis. The unique hydrophobic nature of this large (512 kDa), glycosylated apolipoprotein differs from that of the other apolipoproteins. Since another apolipoprotein, apolipoprotein A-I, has been recently shown to have covalently bound fatty acids, potential fatty acid acylation of apolipoprotein B was investigated. The human hepatoma cell line, Hep G2, synthesizes apoB-100 and secretes the apolipoprotein into the culture medium. After a 24-hr incubation with [14C]palmitate and [14C]stearate, the label was incorporated into apoB-100 when assessed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis, autoradiography, immunoblot analysis, and immunoprecipitation. Hydroxylamine treatment, which hydrolyzes ester and thioester bonds, removed the radiolabel. ApoB-100 isolated from Hep G2 cells by ultracentrifugation and preparative sodium dodecyl sulfate gel electrophoresis was hydrolyzed and analyzed by gas-liquid chromatography-mass spectrometry. In contrast to circulating apoB in low density lipoproteins, both palmitate and stearate were present in newly synthesized apoB-100. These results establish that newly synthesized apoB-100 undergoes covalent acylation with palmitate and stearate. The acylation of apoB may play an important role in lipoprotein particle secretion. In addition, derangements in apoB fatty acid acylation may lead to dyslipoproteinemia.

Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes.

The complete amino acid sequence of PepM49, a peptic fragment of the group A streptococcal type 49 M protein, the antiphagocytic cell surface molecule of the bacteria, is described. This fragment retains the opsonic antibody epitope of the native molecule. The sequence of PepM49, as determined by automated Edman degradations of the uncleaved molecule, and its tryptic and chymotryptic peptides, consists of a total of 143 residues (Mr = 17,187). PepM49, a nephritis-associated M protein serotype, exhibits significant internal homology in its sequence. However, identical sequence repeats of the kind seen in the rheumatic fever-associated serotypes M5, M6, and M24, are absent in PepM49. PepM49 exhibits varying degrees of homology with the M5, M6, and M24 proteins, which is consistent with the existence of variable and conserved regions in the M protein molecule. Predictive analysis as well as CD measurements revealed a high propensity of the PepM49 molecule to assume an alpha-helical conformation. Furthermore, a heptad periodicity of the nonpolar residues, a characteristic of alpha-helical coiled-coil proteins, extends over the entire length of the PepM49 protein. The differences in the nonpolar residue distribution divide the PepM49 sequence into three distinct domains, similar to those seen earlier in the M5 and M6 proteins. Together, these studies establish a conserved conformational design for the sequentially diverse M protein serotypes. However, the pattern of heptad periodicity in the PepM49 protein is quite distinct from that present in the PepM5 and M6 proteins, suggesting distinct differences in structural features among conformationally similar M protein serotypes. This may have relevance to the pathological differences associated with these M protein serotypes.

Human proapolipoprotein A-I: development of an antibody to the propeptide as a probe of apolipoprotein A-I biosynthesis and processing.

In human plasma, apolipoprotein A-I is present as pro and mature isoproteins. The development of a highly specific antibody to the pro isoprotein of apoA-I has been difficult due to the close structural similarity between the pro and mature isoforms of apoA-I. To sermount this difficulty, a peptide was synthesized by the solid phase method which corresponded to the amino acid sequence present in the pro region of apoA-I. The synthetic peptide was coupled to serum albumin and the conjugate utilized to immunize rabbits for antibody production. Immunoblot analysis of purified proapoA-I and mature apoA-I revealed that the antibody was specific for the propeptide of apoA-I. Analysis of apoA-I in the plasma from a Tangier disease patient and newly secreted apoA-I from HepG2 cells clearly demonstrated the isoforms which contained the proisoprotein. The proapoA-I specific antibody should prove to be a useful tool in developing a radioimmunoassay for quantitation of the proisoprotein in plasma, isolation of proapoA-I from normal and dyslipoproteinemic subjects by immunoaffinity chromatography and in studies related to the synthesis and processing of apoA-I.

Acetylated N-terminal structures of class III alcohol dehydrogenases. Differences among the three enzyme classes.

The protein chains of mammalian alcohol dehydrogenases typically lack free alpha-amino groups. The blocked N-terminal regions of the class III type of the rat (ADH-2), human (chi chi) and horse enzymes were isolated by digestions with proteases, and characterized by mass-spectrometry supplemented with chemical analysis of the peptides and their redigestion fragments. Results were confirmed by synthesis of the corresponding peptides, followed by chromatographic comparisons of the native and synthetic products. The N-terminal regions of the three class III alcohol dehydrogenase subunits are homologous but differ from the class I and II enzymes in both the exact start position and the amino acid sequence, which suggests that different N-terminal structures are typical for each of the three classes.

Difference in the structural features of streptococcal M proteins from nephritogenic and rheumatogenic serotypes.

The association of only certain M protein serotypes of group A streptococci with acute glomerulonephritis is very well recognized. Structural information on the M protein, a dimeric alpha-helical coiled-coil molecule, has come so far from three rheumatogenic serotypes, 5, 6, and 24. However, M proteins from the nephritogenic serotypes have not been well characterized. In the present study, we have isolated a biologically active 20,000 Mr pepsin fragment of type 49 M protein (PepM49), a nephritogenic serotype, and purified it to homogeneity using DEAE-Sephadex and gel filtration. The amino acid composition of PepM49 is similar to those of the rheumatogenic M protein serotypes PepM5, PepM6, and PepM24. However, the sequence of the NH2-terminal 60 residues of PepM49 shows little homology to any of these M protein serotypes, although the latter have significant homology among themselves. Nevertheless, PepM49 exhibits a strong heptad periodicity in its nonpolar residues, suggesting its overall conformational similarity with the other M molecules. During the course of the present studies, Moravek et al. (17) reported the NH2-terminal sequence of another M protein serotype, PepM1, which also does not exhibit much homology with the PepM5, PepM6, and PepM24 proteins. Our analysis of this sequence revealed that the PepM1 protein also exhibits a heptad periodicity of the nonpolar amino acids. A closer examination has revealed that the pattern of heptad periodicity in PepM49 and PepM1 proteins is more regular and more similar to each other than has been previously seen for the PepM5, PepM6, and PepM24 proteins. PepM1 is also a nephritogenic serotype. Taken together, these findings indicate an underlying conservation of the tertiary structure of the various M protein serotypes, despite the complexity in their antigenic variation and suggest that the nephritogenic M protein serotypes M1 and M49 may be further apart evolutionarily from the rheumatogenic serotypes 5, 6, and 24. The distinct differences in the structural features of the PepM1 and PepM49 proteins relative to the PepM5, PepM6, and PepM24 proteins are also suggestive of a correlation with the earlier broader classification of the group A streptococci into rheumatogenic and nephritogenic serotypes.