Copolymerization of two distinct tubulin isotypes during microtubule assembly in vitro.

Cells contain multiple tubulin isotypes that are the products of different genes and posttranslational modifications. It has been proposed that tubulin isotypes become segregated into different classes of microtubules each adapted to specific activities and functions. To determine if mixtures of tubulin isotypes segregate into different classes of polymers in vitro, we used immunoelectron microscopy to examine the composition of microtubule copolymers that assembled from mixtures of purified tubulin subunits from chicken brain and erythrocytes, each of which has been shown to exhibit distinct assembly properties in vitro. We observed that (a) the two isotypes coassemble rapidly and efficiently despite the fact that each isotype exhibits its own unique biochemical and assembly properties; (b) at low monomer concentrations the ratio of tubulin isotypes changes along the lengths of elongating copolymers resulting in gradients in immuno-gold labeling; (c) two distinct classes of copolymers each containing a distinct ratio of isotypes assemble simultaneously in the same subunit mixture; and (d) subunits and polymers of different isotypes associate nearly equally well with each other, there being only a slight bias favoring interactions among subunits and polymers of the same isotype. The observations agree with previous studies on the homogeneous distribution of multiple isotypes within cells and suggest that if segregation of isotypes does occur in vivo, it is most likely directed by cell-specific microtubule-associated proteins (MAPs) or specialized intracellular conditions.

Automated enzyme immunoassay for lutropin with the Abbott IMx analyzer.

An automated enzyme immunoassay for human lutropin for use with the Abbott IMx analyzer is described. The assay provides results in approximately 40 min with a sensitivity of 0.25 int. units of LH per liter for up to 23 serum or plasma samples. Cross-reactivity with follitropin (2000 int. units/L) and thyrotropin (2 int. units/L) was negligible; it was 0.016% with human choriogonadotropin (1 X 10(6) int. units/L). There was no interference by high concentrations of bilirubin (0.5 g/L), hemoglobin (7.50 g/L), or triglycerides (13.5 g/L). Intra-, inter-, and total assay CVs were less than or equal to 3.75%, less than or equal to 7.1%, and less than or equal to 7.94%, respectively. Values obtained with the IMx correlated well (r = 0.98, n = 194) with values obtained with Diagnostic Products' LH Double Antibody RIA, and Serono's LH MAIAclone assay. This assay should be useful for small to medium-size laboratories involved in the clinical diagnosis of reproductive pathology.

Effect of cholesterol feeding on lipolytic activities in high- and low-responding rhesus monkeys.

We compared the activities of lipoprotein (LPL) and hepatic lipase (HL) in postheparin plasma in groups of high- and low-responding rhesus monkeys fed a low cholesterol diet followed by a high cholesterol diet. Cholesterol feeding resulted in a two-phase response in lipolytic activities: an initial phase lasting about 21 days that was similar in the two groups, followed by the second phase in which major differences became apparent between the groups. In the initial phase, LPL and HL activities increased along with plasma cholesterol and high density lipoprotein (HDL) cholesterol, but there was no change in plasma triglycerides or apolipoprotein (apo) A-I levels. These changes suggest that both high and low responders respond in a similar manner to metabolize an increased lipid load and that both groups continue to remove triglyceride efficiently while cholesterol accumulation begins. The second phase (between 21 and 42 days and thereafter) in high responders was characterized by a decrease in LPL and HL activities along with plasma HDL cholesterol and apo A-I levels, continued increase in plasma cholesterol, and a slow increase in plasma triglycerides. In low responders, LPL activity was maintained at a high level, HL activity decreased to the basal level, and plasma lipids were unchanged. All changes occurred simultaneously, suggesting metabolic relationships between plasma lipolytic activities, plasma lipids, and apolipoprotein.

Lipoproteins and apolipoproteins in postmortem serum.

In order to correlate lipoprotein and apolipoprotein profiles with atherosclerotic lesions at autopsy, the feasibility of using postmortem serum in these determinations was evaluated. Lipoprotein and apolipoprotein profiles in dogs and monkeys were compared in pre- and postmortem serum samples, and it was found that, up to 24 h postmortem, no appreciable differences were observed in apolipoprotein A-I, B, E, and A-IV serum concentration when compared to premortem values. Electrophoretic mobilities and apolipoprotein gradient gel immunoblotting also revealed no differences in apolipoprotein size distribution. We conclude that postmortem sampling up to 24 h can be used effectively to approximate premortem lipoprotein and apolipoprotein profiles provided that careful sampling techniques are observed.

Effect of ovariectomy on response to dietary cholesterol in patas monkeys (Erythrocebus patas).

From a group of 20 patas monkeys (Erythrocebus patas), four high and four low responders to dietary cholesterol were selected for this study. The monkeys were paired according to their responsiveness to cholesterol, and ovariectomies were carried out on one group of the matched pairs. For the study, the monkeys were fed a Prudent Diet (at 0.1 mg cholesterol/cal) for 5 months preceding the ovariectomy and for 7 months after the ovariectomy followed by a Rich Diet (at 0.4 mg cholesterol/cal) for 7 months. Serum cholesterol, apo B, and apo A-I concentrations and cholesterol distributions were determined. We observed that while the monkeys consumed the Prudent Diet, total serum cholesterol. HDL cholesterol, and apo A-I increased in the ovariectomized group, but not in the control group. When they began consuming the Rich Diet, total serum cholesterol, apo B, and apo A-I concentrations increased in all monkeys, but increased more in the ovariectomized monkeys. The effect of the loss of ovarian function on lipoprotein metabolism is accentuated when the monkeys are fed a Rich Diet designed to be similar to a saturated fat, cholesterol-rich diet consumed by human beings.

The relative contributions of polymer annealing and subunit exchange to microtubule dynamics in vitro.

Microtubules that are free of microtubule-associated protein undergo dynamic changes at steady state, becoming longer but fewer in number with time through a process which was previously assumed to be based entirely on mechanisms of subunit exchange at polymer ends. However, we recently demonstrated that brain and erythrocyte microtubules are capable of joining end-to-end and suggested that polymer annealing may also affect the dynamic behavior of microtubules in vitro (Rothwell, S. W., W. A. Grasser, and D. B. Murphy, 1986, J. Cell Biol. 102:619-627). In the present study, we first show that annealing is a general property of cytoplasmic microtubules and is not a specialized characteristic of erythrocyte microtubules by documenting annealing between tryosinolated and detyrosinolated brain microtubules. We then examine the contributions of polymer annealing and subunit exchange to microtubule dynamics by analyzing the composition and length of individual polymers in a mixture of brain and erythrocyte microtubules by immunoelectron microscopy. In concentrated preparations of short-length microtubules at polymer-mass steady state, annealing was observed to be the principal factor responsible for the increase in polymer length, whereas annealing and subunit exchange contributed about equally to the reduction in microtubule number.

Density gradient characterization of the high density lipoproteins in cholesterol-fed hyper- and hyporesponding patas monkeys (Erythrocebus patas).

Diet-induced changes in high density lipoprotein (HDL) density and size were studied in patas monkeys. When the animals were switched from a moderate fat-low cholesterol diet to a high fat-high cholesterol (HFHC) diet, the plasma apoA-I levels increased initially in all of the animals. The apoA-I levels remained elevated in monkeys able to maintain their plasma cholesterol concentrations near basal levels (hyporesponders), but began to decrease in monkeys who became severely hypercholesterolemic (hyperresponders), reaching levels as low as 65-70% of their basal value by 24 weeks. The larger, lipid-rich HDL (HDL2) was shown by density gradient ultracentrifugation and gradient-PAGE (polyacrylamide gel electrophoresis) to be the HDL fraction responsible for these changes in apoA-I, completely accounting for the increase in apoA-I in hyporesponders and the decrease in apoA-I in hyperresponders. The HDL3 levels remained unchanged in hyporesponders but increased markedly in hyperresponders, partially compensating for the decrease of HDL2 in those animals. Gradient-PAGE showed the HDL3 to be heterogeneous, containing at least two populations of particles of the same density but differing significantly in size. The smaller of these HDL3 were most prominent in the HFHC-fed hyperresponders. These data show that nonhuman primate HDL is both physically and metabolically heterogeneous, and indicate that a high fat-high cholesterol diet-induced hypercholesterolemia severely depresses the HDL2 levels.

Lipoprotein profiles in rhesus monkeys with divergent responses to dietary cholesterol.

From a group of 53 rhesus monkeys, we selected 12 animals, the six with the highest and the six with the lowest response to a high cholesterol diet, and we made detailed analyses of their cholesterol and apolipoprotein profile. The high responders differed from the low responders in several ways. During the high cholesterol diet period, the high responders had much higher plasma apolipoprotein B and E concentrations and much lower plasma apolipoprotein A-I concentrations than did the low responders. Nearly all the increase in plasma cholesterol and apolipoproteins B and E concentrations in the high responders occurred in the lower density fractions (d = 1.006-1.030 g/ml), while the decrease in plasma apolipoprotein A-I concentrations in the high responders was confined to the lower density fraction of the high density lipoproteins (HDL), i.e., HDL2 (d = 1.063-1.125 g/ml). In the low responders, on the other hand, the slight increase in cholesterol concentrations was evenly distributed between the lower density fractions and HDL, and the increase in apolipoprotein A-I concentration of the lower density fractions is related to the decrease in the concentration in the HDL2 in the high responders.

Primary structure of very low density apolipoprotein C-II of human plasma.

Apolipoprotein C-II (apoC-II), a protein constituent of very low density lipoproteins of human plasma and the activator protein of lipoprotein lipase, has been isolated and its amino acid sequence has been studied. The protein has 78 amino acid residues and is lacking cysteine, cystine, and histidine. Chromatography on Bio-Gel P-30 in 25% formic acid of the cyanogen bromide digest of apoC-II yields three fragments designated as CNBr-I, -II, and -III. They contained 50, 19, and 9 residues, respectively. The alignment of the cyanogen bromide fragments has been established as CNBr-III-I-II by isolation and sequence of the tryptic peptides of the intact protein. The amino acid sequences of the tryptic and CNBr peptides were determined by conventional methods. With this information, it was possible to establish the complete amino acid sequence of apoC-II.

Biosynthesis of the dimethylbenzene moiety of riboflavin and dimethylbenzimidazole: evidence for the involvement of C-1 of a pentose as a precursor.

The relative incorporations of specially labeled pyruvate, lactate, erythritol, D-erythrose, D-ribose, and D-glucose precursors into the dimethylbenzene carbon atoms of the 5,6-dimethylbenzimidazole unit of vitamin B12 by Propionibacterium shermanii have been determined. The incorporation data provide information regarding the putative four-carbon biosynthetic unit which is involved in the formation of 6,7-dimethyl-8-ribityllumazine and which is the source of the eight dimethylbenzene carbon atoms of both 5,6-dimethylbenzimidazole and riboflavin. The relative incorporations of the labeled lactate and pyruvate precursors are not consistent with either acetoin or 2,3-butanedione functioning as the four-carbon biosynthetic unit. The relative incorporations of the labeled hexose, pentose, and tetrose precursors indicate that the observed incorporation of C-1 of the pentose into the dimethylbenzene carbon atoms does not involve metabolism to a tetrose intermediate, but occurs more directly. It is concluded that the C-1 position of a pentose precursor is involved in the formation of the putative four-carbon biosynthetic unit.

Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). I. The amino acid sequence of cyanogen bromide fragment II.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is one of the major protein constituents of human plasma high density lipoproteins. The protein has 245 amino acid residues, including 3 residues of methionine, and is lacking isoleucine, cystine, and cysteine. Cleavage of apoLP-Gln-I with cyanogen bromide yields four fragments, designated in their order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the present study, we report the complete amino acid sequence of the NH2-terminal fragment, CNBr II, a peptide that contains 90 amino acid residues.

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). II. The amino acid sequence and alignment of cyanogen bromide fragments IV, III, and I.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is the major protein constituent of the human plasma high density lipoproteins. Cleavage of this protein with cyanogen bromide yields four fragments, designated in the order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the first paper in this series, the amino acid sequence of the NH2-terminal fragment, CNBr II, is reported. In the present study, CNBr IV, III, and I, containing, respectively, 25, 36, and 94 amino acids were sequenced by conventional means. To establish the alignment of the cyanogen bromide fragments, apoLP-Gln-I was digested with trypsin and two of the three methionine-containing tryptic peptides were isolated. The amino acid sequence of apoLP-Gln-I is as follows: (see article). With the complete amino acid sequence available, a CPK space-filling model of apoLP-Gln-I was constructed. The protein was placed into an alpha helical conformation wherever the primary structure permitted. Thirteen helical regions were identified. These regions account for 70% of the amino acid residues of the protein. Each helix is characterized as having two faces (amphipathic). One is a polar face that occupies approximately 180 degrees of the surface of the helix and the other is a hydrophobic face that occupies the other 180 degrees of the helical surface. Similar amphipathic helices have been identified previously in the other lipoprotein-proteins that have known sequences. It is suggested that the amphipathic helical regions of apoLP-Gln-I are important in the binding of phospholipids in high density lipoproteins.

The primary structure of high density apolipoprotein-glutamine-I.

The major protein constituent of human plasma high density lipoproteins has been isolated and its complete amino-acid sequence determined. The protein, designated apolipoprotein-glutamine-I by the presence of carboxyl-terminal glutamine, is a single polypeptide chain of 245 amino-acid residues, including three residues of methionine. The protein is devoid of cysteine, cystine, and isoleucine. Cleavage of apolipoprotein-glutamine-I with cyanogen bromide yields four fragments with 94, 90, 36, and 25 amino acids. The amino-acid sequence of each fragment was determined by conventional methods, with proteolytic digestion with trypsin, chymotrypsin, and thermolysin. The alignment of the cyanogen bromide fragments was determined by the isolation of the methionine-containing tryptic peptides from apolipoprotein-glutamine-I. Inspection of the sequence of apolipoprotein-glutamine-I suggests an interesting distribution of amino acids that may account for its helical structure and its ability to bind and transport lipid.