Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers

On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQwl, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DRI-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1 . In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQwl-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.     

The major type-1 protein phosphatase catalytic subunits are the same gene products in rabbit skeletal muscle and rabbit liver

The catalytic subunit of protein phosphatase-1 (PP1) isolated from rabbit liver had the same electrophoretic mobility as, and yielded peptide maps identical to those of the 33 kDa form of rabbit skeletal muscle PP1. The predicted amino-acid sequences of PP1 obtained from three rabbit liver cDNA clones were identical to that of PP1 alpha from rabbit skeletal muscle. These findings suggest that the distinctive substrate specificities and regulatory properties of hepatic and skeletal muscle type-1 protein phosphatases are not conferred by the catalytic subunits themselves, but by regulatory subunits that are complexed to the catalytic subunits in vivo.     

Multiplicity of heme oxygenase isozymes. HO-1 and HO-2 are different molecular species in rat and rabbit

We report on the detection and characterization of two forms of heme oxygenase in rabbit tissues and provide data suggesting that heme oxygenases in rat and rabbit are not identical and constitute a group of heterogenous proteins. Certain molecular properties, however, are shared by the isozymes in rat and rabbit; the predominant form of the enzyme in control liver and testis is HO-2, in the liver HO-1 is the inducible form, and in the brain HO-1 is not detectable. HO-1 was purified from liver of rabbits treated with bromobenzene to near homogeneity with a specific activity of 8,270 nmol of bilirubin/mg/h and compared with a homogenous preparation of rat HO-1 with a specific activity of 6,220, also obtained from bromobenzene-treated animals. Rat and rabbit HO-1, on sodium dodecyl sulfate-polyacrylamide gel, had molecular weights of 30,000 and 30,700, respectively. Rabbit HO-2 was partially purified from testis to a specific activity of 386 nmol of bilirubin/mg/h and compared with a purified preparation of rat testis HO-2 with a specific activity of 5,700. Using Western immunoblotting, rabbit HO-2 displayed intense cross-reactivity with antibody raised in rabbit to sodium dodecyl sulfate-denatured rat HO-2, and had a substantially larger molecular weight than the rat HO-2 (42,000 versus 36,000). Rabbit HO-1 did not cross-react with antibody to rat HO-1 which was also raised in rabbit. Unlike the rat enzymes, rabbit HO-1 and HO-2 did not differ in thermolability. It is speculated that HO-1 in rat and rabbit, and possibly HO-2, have evolved from divergent evolution of a common ancestral gene(s).     

Separation of two different heavy meromyosins. Evidence for the presence of myosin isozymes in rabbit skeletal muscle.

Heavy meromyosin prepared from rabbit skeletal myosin by chymotryptic digestion was separated into two different heavy meromyosins by Sepharose 4B-6 aminohexyl PPi column chromatography. SDS-gel electrophoresis of one fraction of heavy meromyosin, which was eluted with 75 mM ammonium acetate, showed that it contained the small polypeptide chains, g3 and g2, as well as the large chains. The other fraction of heavy meromyosin, which was eluted with 85 mM ammonium acetate, contained g1 and g2. We concluded that the two heavy meromyosins arose from two different populations (isozymes) of myosin. No significant difference in Ca2+-ATPase activity was detected between the two heavy meromyosins.     

The evolution of alpha-1 and alpha-2-macroglobulin levels in the serum of rabbit fetus and newborn

The study of the evolution of alpha-1 and alpha-2-macroglobulin levels in the rabbit fetus and newborn reveals an analogy between alpha-1-macroglobulin in the rabbit and alpha-2-macroglobulin in humans.     

7alpha- and 7beta-hydroxy-epiandrosterone as substrates and inhibitors for the human 11beta-hydroxysteroid dehydrogenase type 1

The human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes both the NADP(H)-dependent oxido-reduction of cortisol and cortisone and the inter-conversion of 7alpha- and 7beta-hydroxy-dehydroepiandrosterone (DHEA) through a 7-oxo-DHEA intermediate. As shown with human liver and intestine fractions, 7alpha-hydroxy-epiandrosterone (7alpha-hydroxy-EpiA) and 7beta-hydroxy-EpiA were readily inter-converted with no evidence for a 7-oxo-EpiA intermediate. Whether this inter-conversion resulted from action of the 11beta-HSD1 or from an unknown epimerase is unresolved. Furthermore, whether these steroids could inhibit the cortisol-cortisone oxido-reduction remains a question. The recombinant human 11beta-HSD1 was used to test these questions. NADP(+) supplementation only provided the production of 7beta-hydroxy-EpiA out of 7alpha-hydroxy-EpiA with a V(max)/K(M) ratio at 0.1. With NADPH supplementation, both 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA were formed in low amounts from 7beta-hydroxy-EpiA and 7alpha-hydroxy-EpiA, respectively. These inter-conversions occurred without a trace of the putative 7-oxo-EpiA intermediate. In contrast, the 7-oxo-EpiA substrate was efficiently reduced into 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA, with V(max)/K(M) ratios of 23.6 and 5.8, respectively. Competitive and mixed type inhibitions of the 11beta-HSD1-mediated cortisol oxidation were exerted by 7alpha-hydroxy-EpiA and 7beta-hydroxy-EpiA, respectively. The 11beta-HSD1-mediated cortisone reduction was inhibited in a competitive manner by 7-oxo-EpiA. These findings suggest that the active site of the human 11beta-HSD1 may carry out directly the epimeric transformation of 7-hydroxylated EpiA substrates. The low amounts of these steroids in human do not support a physiological importance for modulation of the glucocorticoid status in tissues.     

Differential interactions of rabbit plasma alpha-1-antiproteinases S and F with porcine trypsin

Two major forms of rabbit plasma alpha-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine p-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.     

Studies of esterase 6 in Drosophila melanogaster. XIII. Purification and characterization of the two major isozymes

Esterase-6 (EST 6; carboxylic-ester hydrolase; EC 3.1.1.1) from Drosophila melanogaster was purified to homogenity. Purified enzyme occurs as two closely moving isozymes, slow (EST 6S) and fast (EST 6F), on native polyacrylamide gel electrophoresis. Except for slight differences in their mobility, the two isozymes share similar molecular and catalytic properties. Both isozymes are glycoproteins and have an apparent molecular weight of 62,000 to 65,000 as judged by analytical gel filtration and sodium dodecyl sulfate (SDS) electrophoresis. They have identical mobility on SDS-polyacrylamide gels and an isoelectric point of 4.5. Each isozyme has a single active catalytic site as confirmed by titration with 0,0-diethyl-p-nitrophenyl phosphate (Paraoxon). We conclude that EST 6 is a monomeric enzyme. The amino acid composition of the two isozymes is very similar and both variants lack half-cystine residues. The low pI of the enzyme is due in part to a relatively high proportion of glutamic and aspartic amino acid residues. Characterization of the kinetic parameters of the isozymes using beta-naphthyl and p-nitrophenyl esters revealed no statistically significant differences in catalytic efficiency. There is, however, a suggestion that the two isozymes may differ in their substrate specificity.     

Cloning and sequencing of cDNA coding for rabbit alpha-1-antiproteinase F: amino acid sequence comparison of alpha-1-antiproteinases of six mammals

Rabbit liver cDNA coding for alpha-1-antiproteinase F has been isolated and sequenced. The protein sequence deduced from the nucleotide sequence consists of a 24 amino acid signal peptide and 389 amino acids of the mature polypeptide. Rabbit alpha-1-antiproteinase F showed 74 and 64% homology to human alpha-1-antiproteinase at the nucleotide and amino acid levels, respectively, but the N-terminal five amino acids are lacking in the rabbit protein. The sequences of alpha-1-antiproteinase F of rabbit, human, baboon, sheep, rat, and mouse show about 40% identity, and the reactive site (Met-Ser) is conserved. On the other hand, variable regions are located in the second half to the C-terminal as well as in the N-terminal region.     

Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates.

A method for the assay of neuraminidase in human cultured fibroblasts has been worked out. The substrates, all naturally occurring, were: sialyloligosaccharides (alpha(2 lead to 3)sialyllactose, alpha(2 leads to 6)sialyllactose, disialyllactose), sialylglycoplipids (disialogangliosides GD1a and GD1b), sialylglycoproteins and sialylglycopeptides (ovine submaxillary glycoprotein and its pronase-glycopeptides). The method was based on the determination of the enzymically liberated N-acetylneuraminic acid (NeuAc) by a chromatographic-colorimetric microprocedure. The enzyme acted on sialyloligosaccharides and, in the presence of Triton X-100, on gangliosides, while it did not appreciably affect sialylglycoproteins and sialylglycopeptides. The optimum pH was 4.0 for all tested substrates; the Km values were higher for sialyloligosaccharides (about 10(-3) M), lower for gangliosides (about 10(-4) M); the apparent maximum velocity was higher with alpha(2 leads to 3)sialyllactose (400 mU/mg protein); the reaction rate was linear with time for up to 2 h, and with up to 0.6 mg of enzymic protein. The assay method proved to be sufficiently sensitive (3-4 nmol liberated NeuAc), simple, and reproducible (mean activity on pooled fibroblasts with alpha(2 leads to 3)sialyllactose: 400 mU +/- 6 S.E.).     

Comparison of enzymatic activities of the HIV-1 and HFV integrases to their U5 LTR substrates.

The human immunodeficiency virus type 1 (HIV-1) and human foamy virus (HFV) integrase proteins were overexpressed in Escherichia coli, and purified to a near homogeneity by one- or two-step purification scheme. The endonucleolytic, integration, and disintegration activities for the HIV-1 and HFV integrases were characterized in vitro. The endonucleolytic activities for the HIV-1 and HFV integrases were found only on their own substrates, respectively, indicating that the cognate U5 LTR sequences in the substrates is critical for specific cleavage. However, the integration and disintegration activities showed less specificity on the substrate usage. Our results suggest that the disintegration activity have more preference for substrates based on Y-shaped structure rather than on viral donor DNA sequence.     

Identification of the AMP binding sites of rabbit phosphofructo-1-kinase isozymes B and C

Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo.     

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.     

Polymorphisms of alpha-1-acid (orosomucoid), alpha-2-HS-glycoproteins and alpha-1-B among the Parsis of India.

Genetic polymorphisms of plasma alpha 1-acid glycoprotein (oro-somucoid, ORM), alpha 2-HS-glycoprotein (A2HS) and alpha 1-B-glycoprotein (alpha 1B) were studied in a group of Parsis in Bombay, India. The frequencies of ORM1*1, ORM1*2 and ORM1*3 were found to be 0.636, 0.356 and 0.008, respectively. A2HS*1, A2HS*2 and A2HS*3 frequencies were 0.855, 0.135 and 0.010, while the frequencies of A1B*1 and A1B*2 were 0.881 and 0.119, respectively. The phenotype distribution at all three loci was at Hardy-Weinberg equilibrium. The ORM2 locus was monomorphic in the Parsis.