An enzymatically inactive variant of human lactate dehydrogenase-LDHBGUA-1 Study of subunit interaction

The LDHBGUA-1 variant is an electrophoretic variant which is enzymatically inactive. It is only detectable because of its ability to form heterotetramers with A and/or active B subunits and alter the electrophoretic pattern, although all evidence suggests the B-GUA-1 subunits are always enzymatically inactive. All tetrameric combinatious of active plus inactive subunits including either an A or active B plus three inactive B subunits possess enzymatic activity. The heterotetramers composed of A and B-GUA-1 subunits are more thermostable than A4 homotetramers but less thermostable than normal AB heterotetramers. The AB-GUA-1 heterotetramers composed of active A and inactive B subunits have a Km for pyruvate, for lactate and for NADH which is similar to that observed for normal AB heterotetramers. The substrate specificity of the A plus normal B heterotetramer and the A plus variant B heterotetramer with [alpha]-hydroxybutyrate or the acetylpyridine analog of NAD as substrate are similar, while differences between the normal and variant erythrocyte isozymes are observed when glyoxalate is the substrate. Interaction with the B-GUA-I subunit reduces the sensitivity of the A subunit to urea inhibition (independent of dissociation), as does the active B subunit, but the inactive B subunit does not modulate the inhibition by oxalate. Although a single active subunit in a tetrameric conformation is sufficient for enzymatic activity, many of the kinetic properties of the lactate dehydrogenase molecule reflect the tetrameric structure rather than the sum of independent subunits. Thus communication among the subunits must exist and conformational changes which affect the catalytic properties of the enzyme (-lactate:NAD+ oxidoreductase, EC 1.1.1.27) must occur during tetramer formation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24030/1/0000279.pd

Hominoid triosephosphate isomerase: Characterization of the major cell proliferation specific isozyme

Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35 S ]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [ 35 S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains an blocked NH 2 -terminus and length heterogenity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45351/1/11010_2004_Article_BF00219326.pd

Characterization of two new electrophoretic variants of human triosephosphate isomerase: Stability, kinetic, and immunological properties

Two new electrophoretic variants of human triosephosphate isomerase (TPI) have been partially purified and characterized. The TPI Manchester variant, a cathodally migrating electrophoretic allozyme identified in an individual with the phenotype TPI 1-Manchester, is associated with a normal level of enzyme activity in erythrocytes and normal kinetic properties. It is very thermolabile at 55 and 57° C, although it is not uniquely sensitive to either guanidine-HCl or urea denaturation. The TPI Hiroshima-2 variant is an anodally migrating allozyme (the phenotype of proband is TPI 1-Hiroshima-2) with normal activity and kinetic properties and also normal stability characteristics. It is inactivated less by antisera raised against normal human TPI than either the normal or the Manchester allozyme. Dissociation-reassociation experiments utilizing these allozymes have confirmed that normal human red blood cell TPI isozymes are produced by a sequence of reactions (presumably deamidations) involving alternating subunits.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44141/1/10528_2004_Article_BF00484936.pd

A pilot study of the use of placental cord blood samples in monitoring for mutational events

A pilot study exploring the examination of placental cord blood samples for mutant proteins with one-dimensional electrophoretic techniques is described. Although technical advances are such that the techniques employed in this study are now partially superceded, the practical problems encountered in this study would be typical of any monitoring program of this type. No mutations altering electrophoretic mobility among a battery of 51 different locus products were encountered in a total of 277 747 locus tests. When these data are combined with similar data from other studies, the mutation rate for electromorphs becomes 0.34 x 10-5/locus/generation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27388/1/0000419.pd

Erythrocyte acid phosphatase phenotype and gestational length: no relationship in a sample of 3001 births

Erythrocyte acid phosphatase (ACP1) phenotype was examined in 3001 Caucasian infants born at the University of Michigan Women's Hospital. Contrary to reports from other studies, there was no relationship between the ACP1 phenotype and risk of preterm birth in either the total sample or when the sample was subdivided by sex of infant.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27696/1/0000082.pd

Hominoid triosephosphate isomerase: Regulation of expression of the proliferation specific isozyme

Three primary isoforms of the dimeric glycolytic enzyme, triosephosphate isomerase (TPI; EC 5.3.1.1), are detected in proliferating human cells. The electrophoretically separable isoforms result from the three possible combinations of constitutive subunits and subunits expressed only in proliferating cells. Only a single primary isoform is observed in quiescent cells. The two subunits, which differ by covalent modification (s), are products of the single structural locus for this enzyme. Expression of the proliferation specific subunit (TPI-2) is detected within 6–10 hr following mitogen stimulation of quiescent human cells, requires RNA synthesis and is inhibited by agents which inhibit interleukin 2 expression or function. Only the constitutive subunit (TPI-1) is detected in proliferating cells from nonhominoid primate species. A single class of TPI mRNA, which is increased > 10 fold following stimulation of quiescent cells, is detected on northern blot analysis and S1 nuclease digestion analysis of RNA from quiescent and proliferating human cells. It is similar in size to the TPI mRNA from proliferating cells of the African green monkey, a primate species not expressing TPI-2. Comparison of the structure of the TPI gene from rhesus monkey (nonexpressing species) to the gene from expressing species does not suggest a mechanism for generating TPI-2. Thus, the regulation of the expression of the hominoid restricted, proliferation specific subunit of TPI has been further defined, although the mechanism for generating TPI-2 remains elusive.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45352/1/11010_2004_Article_BF00228282.pd

Development of molecular approaches to estimating germinal mutation rates I. Detection of insertion/deletion/rearrangement variants in the human genome

DNA from 130 individuals was studied with up to 18 (primarily cDNA) probes for the frequency of variants in this initial experiment to determine the feasibility of this approach to screening for germinal gene mutations. This approach, a modification of the usual restriction enzyme mapping strategy, focuses on the detection of insertion/deletion/rearrangement (I/D/R) variants, because the DNA is digested with only two restriction enzymes before transfer to membranes and hybridization with an extensive series of unrelated probes. Some 4000 noncontiguous, independent DNA fragments ("loci"), functional loci, pseudogenes or anonymous fragments, (a total of ~ 77 400 kb) were screened. 19 different classes and 31 copies of presumably I/D/R variants were detected while 4 different classes and 24 individuals exhibiting base substitution variants were observed. 18 of the 19 I/D/R classes were rare variants, that is, each were observed at a frequency, within this population, of less than 0.01; 3 of the base substitution classes existed at polymorphic frequencies and only 1 was a rare variant. 10 of the I/D/R classes, occurring in a total of 18 individuals, were detected with probes which are not known to be associated with repetitive elements. This is a variant frequency for I/D/R variants without known repetitive elements of 0.15 classes and 0.23 copies for each 1000 kb screened; this would extrapolate to 1600 such variant sites in the genome of each individual. Within the context of a mutation screening program, the rare variants, either with or without repetitive elements, would have a higher probability of being de novo mutations than would polymorphic variants; this former group would be the focus of family studies to test for the heritability of the allele (fragment pattern). Sufficient DNA probes are available to screen a significant portion of the human genome for genetic variation and de novo mutations of this type.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27913/1/0000334.pd

Characterization of a series of electrophoretic and enzyme activity variants of human glucose-phosphate isomerase

A total of 3438 cord blood samples were screened for variants of erythrocyte glucose-phosphate isomerase. The five different electrophoretic, three activity/deficiency, and one thermostability variants distributed among 27 unrelated Caucasian families of that population, plus two electrophoretic variants previously described from three Amerindian tribes were subsequently examined for cryptic variation using activity and thermostability criteria. Although thermostability differences were observed between electrophoretic variants, no microheterogeneity within any one class of variant was detected.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47616/1/439_2004_Article_BF00273834.pd

Studies of the purine analog associated modulation of human erythrocyte acid phosphatase activity

The activity of the human erythrocyte acid phosphatase is modulated by a series of structural analogs of purine. The unsubstituted purine base does not affect the enzyme activity. Addition of a substituent at the number six position usually generates an analog which activates the enzyme while similar substitutions at the two position usually generate an inhibitor. Pyrimidines are generally ineffective as modulators while several modifications of the imidazole ring of the purine analogs do not abolish the modulator activity of the purine analog. The level of response to all active analogs is isozyme specific. Differences in apparent relative affinities among the modulators are noted. The modulators with a positive effect on enzyme activity, are effective in the presence of methanol which is more effective than H 2 0 as a phosphate acceptor. These analogs act by enhancing the rate of transfer of phosphate to H 2 O, while decreasing the rate of transfer to methanol. The results suggest that the purine analogs may act by altering the rate of hydrolysis of the phosphoenzyme intermediate by H 2 0 or may change the rate-limiting step in the catalytic mechanism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45350/1/11010_2004_Article_BF00220780.pd

Genetic characterization of Gainj- and Kalam-Speaking peoples of Papua New Guinea

The research presented focuses on genetic variation in the Gainj- and Kalam-speaking peoples of highland Papua New Guinea. The primary data are typings at 51 genetic loci observed on 600 individuals who reside in 21 census units, called parishes. These data are augmented by cultural and demographic information that has also been collected. Parish sizes are small, ranging from 20 to about 200 individuals. Direct Western contact with these people has been occuring only for the past three decades. Although Westernization is currently increasing, we find that much of the traditional settlement pattern and mate exchange system is preserved. There are segregating variants at 27 loci. Four rare variants are initially described: NP 4-Kalam, ADA 6-Kalam, PEPA 3-Kalam, and FUM 2-Kalam. We find evidence for a new Gm haplotype, a; —, that is recessive to all other Gm haplotypes. It occurs at a high enough frequency, f(a;—) = 0.119, to be considered a “private polymorphism.” Average per locus heterozygosity is estimated to be 0.053. This value is not statistically different from levels observed on two modern urban populations. Thus, there is no evidence for a reduced level of genetic variation in these people, despite small parish sizes and a relatively unacculturated social structure.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37632/1/1330700113_ftp.pd