A partially deficient and atypical equine transferrin variant, TF N

A new, partially deficient and phenotypically atypical transferrin variant, TF N, was detected in sera of a number of Finnhorses belonging to one family. The variant was inherited codominantly. In polyacrylamide gel electrophoresis (pH9.0) of sera, variant N appeared as a single weak band migrating slightly faster than the main anodal band of variant M. After immunoblotting or isolation an additional, still weaker, faster band was observed as well as some trace bands. The cathodal component, which is present in other transferrin variants, could not be convincingly proved. The main component of variant N contained four sialic acid residues.     

Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C

In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid.     

Electrophoretic Analysis of Myelin Proteolipid Protein and Its Deacylated Form

Myelin proteolipid protein is known to contain covalently bound fatty acid. To determine the contribution of the fatty acid to the multiple bands observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretic parameters of the proteolipid protein were compared with those of the deacylated form. The relative mobility and proportion of each band, as well as the retardation coefficient and free electrophoretic mobility, were not altered by removal of the fatty acid moiety. Furthermore, the acylated and deacylated forms bound the same amounts of sodium dodecyl sulfate. These data demonstrate that the presence of covalently bound fatty acids does not account for the electrophoretic heterogeneity of the proteolipid.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Specificity of cellular retinol-binding protein in the transfer of retinol to nuclei and chromatin

We have reported previously that cellular retinol-binding protein (CRBP) is able to transfer retinol to specific binding sites in nuclei and chromatin. In this report, we have examined the specificity of the interaction of the protein moiety of retinol-CRBP (R-CRBP) with chromatin and nuclei in the transfer process. We first determined the ability of apo-CRBP, apo-serum retinol-binding protein (RBP), and apo beta-lactoglobulin (BLG), all capable of retinol binding, to compete with R-CRBP in the transfer of retinol to chromatin and nuclei. Apo-CRBP was an effective competitor but apo-RBP and apo-BLG showed no competitive ability. On the other hand, cellular retinol-binding protein type II (CRBP(II], whose amino acid sequence shows a considerable similarity to CRBP, did compete for the transfer of retinol from the R-CRBP complex, but less effectively than CRBP. These results demonstrate that the interaction of the protein moiety of the R-CRBP complex with nuclei and chromatin is quite specific.     

An electrophoretic analysis of selected enzymes of the coconut crab, Birgus latro.

1. Employing starch gel electrophoresis at pH 8.6, a single anodal band of L(+)-lactate dehydrogenase activity was found in skeletal muscle of Birgus latro. 2. Two anodal malate dehydrogenase isozymes were observed in heart, skeletal muscle and pericardial gland tissue. Lung and gill exhibited the faster moving band only. 3. Multiple bands of esterase activity were detected in all tissues examined employing alpha-naphthylacetate or alpha-butyrate as substrate. 4. Multiple molecular forms of superoxide dismutase were observed in all tissues examined. 5. Lung exhibited a single cathodal band of carbonic anhydrase activity.     

Acid Lipase Inhibitor in Chicken Plasma Identified as Apolipoprotein A-I

We have reported a inhibitor of acid lipases in liver lysosomes and erythrocytes from chickens [M. Fujii et al., Int. J. Biochem., 22, 895–898 (1990)]. In this paper, the properties of the inhibitor were described in comparison with those of apo A-I of chicken.

The purified inhibitor migrated with the same mobility on SDS–PAGE as apo A-I, and had a molecular weight of 27,000. The peptide map from the lipase inhibitor was similar to that of apo A-I. Antibodies to the acid lipase inhibitor also reacted with apo A-I. Apo A-I inhibited the acid lipase activities of liver lysosomes and erythrocytes from chickens as strongly as the lipase inhibitor. The N-terminal amino acid sequence of lipase inhibitor was identical to that of apo A-I as far as residue 20. The amino acid sequence of peptides obtained from the inhibitor by cleavage with CNBr corresponded to internal sequence of apo A-I, and so the CNBr-peptides were derived by cleavage after the methionine residues in apo A-I. The findings showed that the inhibitor of the acid lipases in liver lysosomes and erythrocytes from chickens was identical to apo A-I.     

Purification and crystallization of a retinoic acid-binding protein from rat epididymis. Identity with the major androgen-dependent epididymal proteins

The retinoic acid binding activity in the lumen of the rat epididymis (Ong, D., and Chytil, F. (1988) Arch. Biochem. Biophys. 267, 474-478) has been purified to homogeneity. The protein exists in two forms, one form having an additional three amino acids at the amino terminus. The amino acid sequence of the protein was determined to 20 amino acids and proved to be identical to that of the major androgen-dependent proteins from rat epididymis as deduced from the cDNA sequence. These proteins are thought to play a role in sperm maturation, perhaps, it can be suggested now, by delivering retinoic acid to the sperm. The retinoic acid-binding protein has sequence homology to the serum retinol-binding protein and is predicted to have the same overall fold of the polypeptide chain. The epididymal retinoic acid-binding protein has been crystallized from 39 to 43% saturated ammonium sulfate, 10 mm Tris, pH 8.0. The crystals are space group P2(1), with a = 39.4, b = 58.9, c = 65.4 a, beta = 105 degrees 16 min.     

Expression and genetics of caprine haemoglobins

The expression of haemoglobin (Hb) has been studied in 260 Norwegian Dairy goats by the Immobiline technique at pH ranges 6.7-7.7, 6.9-7.6 and 6.9-7.5. The majority of goats exhibited two- or four-band patterns. In two-band types the average ratio between the anodal and cathodal band was 74:26. PAGE with 8M urea distinguished three phenotypes for the beta chains, proving that the Hb variation described is in the beta chain. Segregation data in 106 complete sire-dam-offspring families agreed with the existence of four beta globin alleles--A2, A4, A6 and A8. Twenty-seven animals had reversed ratios (R) of Hb bands. In two-band phenotypes the average ratio was 36:64. In 15 complete families where one of the parents had reversed ratio, eight offspring received the R type, indicating a simple genetic control. After urea PAGE the R animals all showed the same alpha chain phenotype which differed from that of goats having common ratios of bands. An additional polymorphism appeared in nine animals as three- and five-band patterns which is assumed to be the result of heterozygosity for II alpha and for II alpha and beta globin genes respectively.     

Purification,cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-11: comparison to the human sequence

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-11. Sequence analysis indicated that the monkey apo C-11 cDNA was 200 by longer than the human and the difference in size was all in the 5° untranslated region of the mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-11 cDNA sequence encoding a preprotein of 101 amino acids — identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-11 amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-11 and this is confirmed by isoelectric focusing and immunoblotting.     

alpha-L-fucosidase polymorphism in human semen, blood, and vaginal fluid

The diallelic enzyme alpha-L-fucosidase has been found to be highly active in semen. Eight distinct enzyme bands were observed in each homozygote and the heterozygote consisted of a combination of bands. The isoelectric points ranged between pI 4.34 and 6.65. Activity was found to be much lower in vaginal fluid, not more than three cathodal bands were visible in any sample examined. In leucocytes, 5 bands were detected in homozygotes whilst in serum only two faint anodal bands could be detected. Activity was low in vaginal fluid and could not be detected in urine or saliva. Population studies were carried out on semen samples from Oxford and Basingstoke and on lymphocyte samples from Oxford. The data were in agreement with Hardy-Weinburg equilibrium. The common allele (Fu 1) had a frequency of 0.704 in Oxford semen samples.     

Proteolytic cleavages in alpha 1-antitrypsin and microheterogeneity

Antitrypsin was resolved into two pools by ion-exchange chromatography. Pool 2 contained three anodal isoinhibitors and an N-terminal sequence identical with the one found by others. Pool 1 contained, in addition to the anodal ones two cathodal isoinhibitors as well. The sequencing data of Pool 1 indicate that the cathodal proteins are formed from the anodals by a cleavage of the Gly5-Asp6 bond in the molecule.     

Partial preparative separation and properties of the isoinhibitors of human alpha 1-antitrypsin (alpha 1-protease inhibitor).

Human lapha 1-antitrypsin (alpha 1-protease inhibitor) was chromatographed on a DEAE-cellulose column at pH 6.4. After elution with a linearly increasing concentration of NaCl, five pools (pools I-V) were formed from the eluate, pool I corresponding to the lowest and pool V to the highest concentration of salt. As demonstrated by analytical isoelectric focusing, with increasing concentrations of NaCl the concentration of the cathodal isoinhibitors gradually decreased and the concentration of the anodal ones increased in the pools. Pool I contained only three cathodal and pool V only three anodal isoinhibitors with a limited overlap between the pools. In contrast with the isoinhibitor composition, the sialic acid contents of the pools did not vary with the elution conditions. In line with the chemical evidence, desialylation of the fractions did not affect their electrofocusing positions relative to one another and did not abolish the microheterogeneity of the protein.     

Creatine kinase isoenzyme associated with synaptosomal membrane and synaptic vesicles

Brain creatine kinase is principally of soluble cytoplasmic origin (anodal electrophoretic mobility). However, synaptosomal membranes and synaptic vesicles are enriched in an isoenzyme electrophoretically similar to muscle type creatine kinase (cathodal electrophoretic mobility), but which can be distinguished from muscle type by other means.     

Identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney.

The chaperonin family of proteins, which includes GroEL protein of E. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant chloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial P1 protein. We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA. Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand. After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer. Analysis of column fractions on a 7.5% polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da. Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence. These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support.     

Unusual sialilation of three different rare genetic variants of serum DBP: Gc 1A17, Gc 1A16, and Gc 1A11

Summary The proteins of three anodal Gc1 variants, Gc 1A16, 1A11, and 1A17, are characterized by the most acidic isoelectric points observed so far among the different Gc mutants. Stepwise removal of N-acetylneuraminic acid (NANA) by treatment with neuraminidase was performed to estimate the degree of sialilation of these Gc variants. The results indicate that both proteins, the anodal and the cathodal component of these Gc 1 mutants, carry sialic acid residues. This observation is remarkable in so far as usually only the anodal component of the Gc 1 protein contains NANA and only a single residue. From the experiments carried out it can be deduced that Gc 1A16 has two NANA residues in the anodal and one NANA residue in the cathodal component. Gc 1A16 was found in four members of three generations in a Danish family; the variant segregated as a Mendelian trait. More difficult to interprete are the results obtained with the variants Gc 1A11 and Gc 1A17. Gc 1A11 probably has three NANA residues in the anodal and two NANA residues in the cathodal component. Gc 1A11 has been observed in two mother-child pairs and is presumably also a simple genetic trait. Gc 1A17 has also several NANA residues in both Gc proteins; it is suggested that the anodal component has either three or four NANA residues and the cathodal component either two or three NANA residues. Family information on this variant is not yet available.     

Human thyroxine-binding globulin (TBG): Heterogeneity within individuals and among individuals demonstrated by isoelectric focusing

Isoelectric focusing of human plasma samples labeled in vitro with [¹²⁵I]-thyroxine reveals considerable biochemical and genetic variation in thyroxine-binding globulin. (1) In all individuals tested, at least three primary isoelectric bands are seen in the pH range of 4.2 to 4.5, with additional bands at lower pH ranges. Similar patterns are produced by plasma from nonhuman primates. These band differences appear to be the result of differences in sialic acid content. TBG produces a single electrophoretic band on standard polyacrylamide gel electrophoresis. (2) Genetically determined, X-linked differences in electrophoretic mobility of TBG are observed in several human populations. Female homozygotes or male hemizygotes for the TBG slow variant (TBG-S) produce band patterns shifted by 0.5 pH unit cathodal to the common pattern (TBG-C). Female heterozygotes produce patterns with six-plus bands, representing the simple sum of the common and slow types. This difference is not the result of differences in sialic acid content. The gene frequency of this variant is 10% in American Blacks. (3) In pregnant women additional anodal bands are observed, giving the impression of a shift, by integral steps, in the pattern relative to the nonpregnant type. This shift is abolished by mild neuraminidase treatment.This work was supported by a grant from the O'Brien family of Houston, Texas.     

Interchain disulfide bonds in crotamine self-association

Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.