Apolipoprotein A-I variants. Naturally occurring substitutions of proline residues affect plasma concentration of apolipoprotein A-I.

Six unrelated families with genetically determined structural variants of apo A-I were found in the course of an electrophoretic screening program for apo A-I variants in dried blood samples of newborns. The following structural variations were identified by the combined use of HPLC, time-of-flight secondary ion mass spectrometry (TOF-SIMS), and automated gas phase sequencing: Pro3----Arg (1x), Pro4----Arg (1x), and Pro165----Arg (4x). All variant carriers were heterozygous for their mutant of apo A-I. Subjects heterozygous for apo A-I(Pro165----Arg) (n = 12) were found to exhibit lower mean values for apo A-I (109 +/- 16 mg/dl) and HDL cholesterol (37 +/- 9 mg/dl) than unaffected family members (n = 9): 176 +/- 41 and 64 +/- 18 mg/dl, respectively (P less than 0.001). In 9 of 12 apo A-I(Pro165----Arg) variant carriers the concentrations of apo A-I were below the fifth percentile of sex-matched controls. By two-dimensional immunoelectrophoresis as well as by densitometry the relative concentration of the variant apo A-I in heterozygous carriers of apo A-I(Pro165----Arg) was determined to account for only 30% of the total plasma apo A-I mass instead of the expected 50%. Thus, the observed apo A-I deficiency may be largely a consequence of the decreased concentration of the variant apo A-I. In the case of the apo A-I(Pro3----Arg) mutant, densitometry of HDL apolipoproteins demonstrated a distinctly increased concentration of the variant proapo A-I relative to normal proapo A-I. This phenomenon was not observed in the apo A-I(Pro4----Arg) mutant or in other mutants. This suggests that the interspecies conserved proline residue in position 3 of mature apo A-I is functionally important for the regular enzymatic conversion of proapo A-I to mature apo A-I.

Characterization of a basic transthyretin variant--TTR Arg 102--in the German population.

A basic transthyretin (TTR) variant, apparently non-pathogenic, has been reported in a German family. Protein analysis of this TTR variant revealed the substitution of arginine for proline at position 102 of the TTR polypeptide chain. This result was confirmed by DNA analysis of PCR amplified DNA.

Thyroxine binding in a TTR Met 119 kindred.

Recently, a transthyretin variant, TTR Met 119, in which methionine substitutes for threonine 119, a component of the protein's iodothyronine binding site, was identified in individuals with transient euthyroid hyperthyroxinemia. Healthy carriers of Met 119 have normal serum thyroid hormone concentrations, but two studies of Met 119 carriers have differed as to whether T4 binding to TTR is increased. An additional kindred has been identified by hybrid isoelectric focusing in an ongoing screening program for TTR variants in the Portuguese population with TTR Met 30 associated familial amyloidotic polyneuropathy. Cyanogen bromide peptide mapping and DNA restriction length polymorphism analyses showed that the propositus was a compound heterozygote for two TTR variants: Asn 90 and Met 119. Family analysis revealed that he inherited the TTR Met 119 variant from the mother and the TTR Asn 90 variant from the father. Neither the compound heterozygote nor his parents had symptoms of familial amyloidotic polyneuropathy. Serum dialysis with stepwise saturation of iodothyronine binding sites confirmed that TTR binding of T4 is increased in TTR Met 119. The increased binding is due to a higher TTR concentration rather than an increased association constant for T4. Because of the small proportion of serum T4 bound by TTR, increased T4 binding by TTR did not affect the ratio of free to bound T4 or T4 concentrations. In contrast, plasma retinol binding protein, almost all of which is bound by TTR, was elevated. The Asn 90 mutation does not affect either the concentration or the hormone binding characteristics of the protein. Possible long-term effects of these mutations and the combined heterozygotic state remain to be determined.

Transthyretin Val71Ala mutation in a Dutch family with familial amyloidotic polyneuropathy.

A Dutch family with familial amyloidotic polyneuropathy associated with the transthyretin mutation Val71Ala is described. This is the third reported family with this mutation, causing at the protein level an unstable TTR monomer and at the clinical level progressive wasting, polyneuropathy, autonomic dysfunction and vitreous opacities.

Apolipoprotein E type 4 allele and Alzheimer's disease: effect on age at onset and relative risk in different age groups.

The effect of the apolipoprotein E (apoE) genotype on the age at onset of Alzheimer's disease (AD) and the relative risk conferred by the apoE epsilon 4 allele were studied in 91 patients and 69 healthy age-matched controls. According to the age of presentation, which varied from 44 to 95 years, subjects were divided into four groups. The inheritance of at least one epsilon 4 allele was associated with a significant reduction of the age at onset by 7.7 years among patients who were 83 years or older when examined. A weaker inverse relationship between the epsilon 4 allele and the age at onset was also observed among patients who were aged 44-63 years at presentation. The effect of the epsilon 4 allele was minimal or absent in the two intermediate age categories. The relative risk of AD conferred by the inheritance of at least one epsilon 4 allele showed no consistent age-related pattern. The overall risk expressed as an odds ratio was 5.0 (95% CI 2.4-10.5). With respect to the limitations of the study, we tentatively conclude (1) that the effect of the apoE epsilon 4 allele on the age at onset is not restricted to AD patients of a particular age, in accordance with current hypotheses on the role of apoE gene products in the biology of AD; (2) that the relative risk of AD associated with the epsilon 4 allele is not significantly modulated by age. Although the apoE epsilon 4 allele is an important susceptibility factor for AD occurring in middle age as well as in later life, it is of limited value in routine clinical diagnosis and should not be used for predictive testing in asymptomatic individuals.

IPGMAKER: a program for IBM-compatible personal computers to create and test recipes for immobilized pH gradients.

The program "IPGMAKER" is a computational aid for creating and testing recipes for near-linear immobilized pH gradients. It was written for fast IBM personal computers (with a Type 80386 processor and 80387 coprocessor) and compatibles equipped with a VGA, EGA or Hercules (mono) graphics card. The program is limited to the use of up to 10 acids and/or bases, and to ranges spanning between pH 2 and 12. The resulting recipes are presented either as final concentrations in the 2 chambers of a mixing device for linear gradients or as volumes from 0.2 moles/L stock solutions adjusted to a user-defined average buffering power. One of the subroutines determines the pH, gradient slope and buffering capacity at any location of the gradient and includes a facility to estimate the pI of proteins from the composition of their primary structure.

A new sample applicator for isoelectric focusing in horizontal polyacrylamide gels.

A new sample applicator for horizontal flat gels is described. The applicator is practically safe against contamination from adjacent samples and can be used for all types of electrophoretic separations including a concentration step for either the sample (i.e. disc electrophoresis) or the separated zones (i.e. isoelectric focusing). The applicator is a piece of flat glass with 26 or 51 parallel 2 mm wide grooves, drilled at distances of 9 or 4.5 mm. Samples, maximally 25 or 50, are applied to the areas between the grooves. By inverting the applicator, the samples are brought into close vicinity to the gel surface and the pendant droplets expand by capillary attraction into the slits between the glass and gel with resultant even distribution across the lanes of 2.5 or 7 mm width. The applicator can be used for separations with and without protection of the electrophoretic setup by paraffin oil and allows for fast multiple handling of samples by means of appropriate syringes and microtiter plates.

Pouring reproducible gradients in gels under computer control: new devices for simultaneous delivery of two independent gradients, for more flexible slope and pH range of immobilized pH gradients.

Recently we described (Electrophoresis 5: 143-147, 1984) a new device for producing density and solute gradients, involving computer-controlled coöperation of stepmotor-driven high-precision burettes, the purpose being to substantially improve reproducibility, flexibility, and documentation of gradients used in gels and in other applications in biochemistry. Here we present the functional principle of three modified and partly alternative devices, based on simultaneous delivery of a density gradient and a non-density gradient. They provide unlimited flexibility of choice for the slope of the non-density gradient, which is stabilized by the density gradient. They seem especially useful for pouring immobilized pH gradients in gels of wide pH ranges (between pH 3.5 and 10) where localized flattening of the gradient is needed to selectively improve resolution. Used in two-dimensional electrophoretic analysis, they should considerably improve control of spot coördinates, standardization of the technique, and interlaboratory data exchange.

Potential treatment of transthyretin-type amyloidoses by sulfite.

Familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA) are characterized by systemic extracellular deposition of insoluble transthyretin (TTR) fibrils. While only normal TTR is found in fibrils from SSA patients who predominantly suffer from cardiomyopathy, autosomal dominant FAP preferentially affects peripheral nerves and heart and is associated with so-called amyloidogenic mutations of this protein, giving rise to TTR forms of decreased stability. Using isoelectric focusing in urea gradients we were able to demonstrate a stabilizing effect of sulfite on TTR monomers and tetramers, as well as an increase in the tetramer/monomer ratio. We demonstrate that this ratio, which is decreased in FAP patients, can be increased to beyond normal levels. We show that doses of sulfite which are tolerable in vivo produce a significant increase in the tetramer/monomer ratio and postulate that sulfite may be a potent drug for delaying the onset and progress of FAP and SSA.

Pouring wide-range immobilized pH gradient gels with a window of extremely flattened slope.

A gradient mixing device has been designed to pour polyacrylamide gels with a wide-range immobilized pH gradient including a "window" of extremely flattened slope. The device consists of an IBM-compatible personal computer controlling 8 step-motor-driven burettes, with four producing a density gradient from glycerol and delivering acrylamides and catalysts for gel polymerization, two delivering Immobilines for a wide-range pH gradient and the other two burettes responsible for the delivery of Immobilines for a partial pH range inside the wide range. The effect on a complex separation pattern of proteins with a wide range of pI is that resolution can be increased reproducibly to any reasonable extent at any location of the separation pattern.

Double one-dimensional electrophoresis of human serum transferrin: a new high-resolution screening method for genetically determined variation.

With one-dimensional electrophoresis or electrofocusing the selective demonstration of proteins and their genetically determined variants from complex compositions like the physiological body fluids often is possible only with specific staining techniques or immunofixation. By the method described here samples are separated by PAG electrophoresis and a selected detail of the electropherogram is further resolved by PAG electrofocusing. The resulting pattern of bands after a usual protein stain can easily be interpreted due to the reduction of the number of bands. Informative bands hidden in a one-dimensional pherogram by overlapping with one or more other bands can be selectively visualized by the one-dimensional combination of two electrophoretic methods. With the double one-dimensional technique several hundred samples can be analyzed per person day. Thus the application in electrophoretic screening studies appears to be of special interest. The method is demonstrated by using the polymorphism of human serum transferrin as an example. A new variant of Tf-C with a high incidence in the German population is demonstrated.

Improved screening test for abnormal hemoglobins from dried blood samples.

A method is described wherein blood samples taken from adults or newborns and dried on filter paper can be used for hemoglobin analysis within 2 years after sampling. The samples are eluted in 8 M urea in the presence of 5% 2-mercaptoethanol and 2% of the neutral detergent Nonidet P-40. Then the individual alpha, beta, gamma, and epsilon chains are separated by means of electrofocusing in 8 M urea-PAA gels. Up to 96 samples can be applied to a gel using multiple syringes. Several hundred samples can be analyzed daily by one person. This method may be especially useful for preventive programs against sickle cell anemia as well as for human mutation monitoring systems.

Isoelectric focusing of basic proteins: the problem of oxidation of cysteines.

Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314-325). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e.g. more than 4 h at greater than or equal to 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2-mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2-ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.

Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients.

Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal dominant disease familial amyloidotic polyneuropathy (FAP). Some three quarters of known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoretic procedure sensitive to differences in the stability of tetramers and monomers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 electrically silent mutants of TTR among 2000 plasma samples from German donors. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as due to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric conformations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.

Demonstration of a factor in the serum of homozygotes and heterozygotes for cystic fibrosis by a non-biological technique.

A factor has been isolated from serum of homozygotes and obligate heterozgotes for cystic fibrosis using isoelectric focusing and disc electrophoresis as analytical methods. The factor is focused within an IgG-fraction with an isoelectric point of pH 8 to 9 but differs from IgG in its lower molecular weight. It is thus similar to, if not identical with, the ciliary dyskinesia factor.