Biochemical and genetic studies on rabbit hemoglobin. I. Electrophoretic polymorphism of theβ chain

A genetic polymorphism of beta-chain rabbit (Oryctolagus cuniculus) hemoglobin is demonstrated by means of acid starch gel electrophoresis. The biochemical evidence presented suggests that a previously reported substitution of a neutral amino acid for a histidine is responsible for the detected genetic variation. Segregation analysis was performed in a sample of 15 matings with 49 offspring and confirmed the genetic hypothesis: two common alleles at an autosomal locus. The calculated gene frequencies in a random sample of 125 individuals are HBB*1 = 0.48 and HBB*2 = 0.52.     

Comparative studies on the activity and polymorphism of enzymes in the semen of farm animals and birds—I. Electrophoretic investigation into the non-specific esterases in bull seminal plasma

• 1.1. Starch gel electrophoresis was carried out on the non-specific esterases in bull seminal plasma.
• 2.2. Differentiation of non-specific esterases was performed by using different substrates and inhibitors.
• 3.3. It is suggested that the non-specific esterases in bull seminal plasma belong to the types A, B, C.

     

Biochemical studies of the chromaffin granule. II. Properties of membrane-bound and water-soluble forms of chromogranin A and dopamine-β-hydroxylase activity

1. Chromogranin A has been isolated from the washed membrane fraction of highly purified chromaffin granules after solubilization in Triton X-100. A hydrated molecular weight of 2.9·10⁵ has been obtained for chromogranin A_M which in its electrophoretic mobility and molecular volume appears very similar to chromogranin A_I of the water-soluble fraction.     

Rabbit hemoglobin: The heterogeneity of the β-chain

Two new allelomorphs of rabbit hemoglobin βT5 peptide are described. One variant contains glutamine and asparagine while the other contains serine and histidine. Both variants can be separated on ion-exchange resin with a pyridine-acetic acid as well as a pyridine-formic acid gradient.     

Biochemical studies on insect haemolymph—II: The nature of the reducing material present

The material in Schistocerca gregaria haemolymph which gives rise to glucose on acidic hydrolysis is shown to be trehalose, of which a crystalline sample was isolated. Glucose and trehalose were also shown to be present in the haemolymph of the larvae of the large cabbage white, Pieris brassicae. In the locust only a relatively small amount of material is present which behaves on assay as glycogen, although it does not appear to be true glycogen. It is shown that amino acids make a significant contribution to the non-fermentable reducing power of the haemolymph as measured by the Hagedorn-Jensen method, and that tyrosine may account for a large part of that contribution.     

Immunogenetic study on the polymorphism of serum α2-lipoproteins in mink. II. Identification of allotypes Lpm-7 and Lpm-8 and genetic control of seven markers of the Lpm system

By means of alloimmunization of mink, two new antigens, Lpm-7 and Lpm-8, were detected in their sera. Lpm-7 and Lpm-8 allospecificities were referred to a very high density ₂-lipoprotein (Lpm) by the following criteria: histochemical tests, immunoelectrophoresis, preparative ultracentrifugation, and coalescence of alloprecipitates with heteroprecipitates in double diffusion tests. Genetic analysis indicated that Lpm-7 and Lpm-8, together with the earlier described Lpm-1, Lpm-2, Lpm-3, Lpm-4, and Lpm-5, share a common immunogenetic system. Polymorphism for the seven markers is conditioned by the genetic units Lpm ⁸, Lpm ⁴, Lpm ^{4, 8}, Lpm ^{4, 7}, Lpm ^{3, 4, 8}, Lpm ^{1, 8}, Lpm ^{1, 2, 7}, and Lpm ^{2, 4, 5, 7}, which behave as alleles. Of these units, the latter six are probably haploid sets of closely linked genes.     

Biochemical characterization of the β-chain variant haptoglobin Marburg

Summary -chains were isolated from two individuals heterozygous for the -chain mutant haptoglobin Marburg. Total amino acid composition and tryptic peptides were compared with -chains from common haptoglobin types. ^Mb chain preparations are characterized by the presence of -chains with an atypical electrophoretic migration rate and by at least three, possibly four additional peptides in their tryptic digests. It is probable that haptoglobin Marburg is the result of an mutational event other than a single base substitution.Supported by US-PHS grant AM 11796 and by US-PHS grant HD 03321 and aided by a grant from the Deutsche Forschungsgemeinschaft.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney.

A study has been made to determine whether renal plasma membranes contain an HCO3 stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney. The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase. The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity. These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Experiments with β-chain variants of the hemoglobin of Mus musculus

Starch gel electrophoretic and ultracentrifuge methods failed to demonstrate any differences between the hemoglobins of mice of the Shanghai and HBBP/Cag strains and crosses among these strains. The apparent identity of these hemoglobins is thought to stem from the contribution of Asian mice to the British mouse fancy from which the laboratory strains having Hbb-p in part descerd. Maleate buffer of pH 7 or above can be used to prevent the formation of disulfide-bridged dimers of mouse hemoglobins. However, the minor electrophoretic bands of Hbb-p and Hbb-d react with approximately twice as much maleate as the major bands of each of these hemoglobins, although the minor bands like the major contain only one free cysteine group per chain. This can be explained by the alkylation of the -amino of lysine residue 76, but some evidence for the alkylation of histidine in the minor band of Hbb-p is also presented.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney

A study has been made to determine whether renal plasma membranes contain an HCO₃^? stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney.The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase.The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome $\text{c}$ oxidase activity.These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Population studies on the inter-α-trypsin inhibitor (ITI) polymorphism

Serum samples from four European populations — Germans from Jena, Germans from Hannover, Italians from Udine, and Slovakians from Bratislava — were tested for the inter-α-trypsin inhibitor (ITI) polymorphism. The results are compared with those reported for other European, Asiatic and African populations. Reviewing all the hitherto published ITI allele frequencies one can see that the variability among European Populations seems to be rather small, whereas Asiatic and African populations are striking by quite different distribution patterns.     

Biochemical and genetic studies of four patients with pyruvate dehydrogenase E1α deficiency

We report studies of four patients with pyruvate dehydrogenase complex (PDH) deficiency caused by mutations in the E1α subunit. Two unrelated male patients presented with Leigh syndrome and a R263G missense mutation in exon 8. This mutation has previously been described in males with the same phenotype. The two other patients had different novel mutations: (1) an 8-bp deletion at the C-terminus (exon 11) was found in one allele of a young girl suffering from microcephaly and (2) a C88S missense mutation (exon 3) in a boy who only presented with motor neuropathy. These mutations were not found in the mothers of any of the four cases. Immunoblot analysis revealed decreased immunoreactivity for the E1α and E1β subunits in three out of the four patients. These findings confirm that: (1) PDH deficiencies are genetically heterogeneous, (2) the R263G mutation is more frequent in male cases than are other mutations and this amino acid is a hot spot for gene mutations, (3) the last eight amino acids may be important for the conformation of the tetrameric E1-PDH enzyme, and (4) the amino acids at positions 88, 263 and 382–387 are essential for the linking of the α subunit with the β subunit and for the activity of the holoenzyme. Received: 28 October 1996 / Revised: 13 January 1997     

Immunogenetic study on the polymorphism of serum α2-lipoproteins in mink. I. Identification and genetic control of five Lpm allotypes

Five allotypes, Lpm 1, Lpm 2, Lpm 3, Lpm 4, and Lpm 5, were detected by isoimmunization in mink sera. Immunoelectrophoresis, preparative ultracentrifugation, and histochemical tests for lipids and esterase permitted reference of these alloantigenic markers to a very high density ₂-lipoprotein. Based on population analysis and breeding tests, five genetic units are postulated: Lpm ¹, Lpm⁴, Lpm^3,4, Lpm^1,2, and Lpm ^2,4,5. These units determine the polymorphism of the Lpm system and behave as Mendelian alleles.     

Biochemical studies on insect haemolymph—I. Variations in reducing power with age and the effect of diet

The variation with age of eight reducing values of the haemolymph of Schistocerca gregaria was determined both for locusts reared entirely on grass and for those reared on an artificial diet to which a very small quantity of grass had been added. Random variation in results found in the former group was considerably reduced by the use of artificial diet, although in each case the normal patterns of variation with age were quite clear. Measurements of the specific gravity of the haemolymph of S. gregaria, together with Lee's values for the variation in blood volume with age, and the above reducing values, have enabled a calculation to be made of the variation with age of the total amounts of various reducing materials present in locust haemolymph. In particular, a very considerable increase in the trehalose content and a much smaller increase in glucose were shown to occur during the fifth instar.     

Hemoglobin binding with haptoglobin: Delineation of the haptoglobin binding site on the α-chain of human hemoglobin

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the -chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues 121–135. The present study describes a precise delineation of this Hp-binding site on the -chain. Two overlapping peptides (111–125 and 121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (119–135, 119–133, 119–131, 119–129, and 119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of¹²⁵I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide 119–127 retained a Hp-binding activity equivalent to that of peptide 121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (122–127, 121–127, 120–127, 119–127, 118–127, 117–127, 116–127, and 115–127). The binding of¹²⁵I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the -chain of human Hb comprises residues 121–127.     

Experimental studies on the benthic phases of haptophyceae II. studies on ‘Aged’ benthic phases of Pleurochrysis scherffelii E. G. Pringsh

A study has been made of the ‘recovery’ processes of benthic cells of Pleurochrysis scherffelii ‘aged’ in two year-old cultures. Water-line non-motile cells and those from the bottom of the flasks were utilized. The water-line material showed a reduction in cell size and contents, whereas cells from the flask bottom showed an increase in cell diameter with reduction in chromatophore size and increase in the volume of the leucosin reserve, and in the number of lipid vesicles. Both types of nonmotile phase failed to produce motile cells when transferred to higher temperatures and brighter illumination whilst still in the ‘aged’ medium. Transference of the aged benthic cells from the bottom of the flask to ‘enriched’ sea water, to fresh sea water without enrichment, to the aged medium enriched with nitrate-N, phosphate-P and trace elements, or to fresh sea water enriched with soil extract, resulted in the onset of cell division and release of motile cells within 12–24 h, with the general indication that micronutrients, organic or inorganic, were responsible for the ‘pace’ of cell division. Under similar conditions, the aged water-line cells showed cell division when transferred to fresh enriched media, but no release of motile cells, and invariably death of the mass of daughter cells followed the initial rapid proliferation. In aged media of different biological history, the ‘young’ benthic cells grown on sea water agar showed further cell division, but with the development of different morphological forms from those obtained in the aged ‘Pleurochrysis’ medium, and without release of motile cells. Short-term immersions of the aged benthic cells from the flask bottom in enriched media (for 1, 3, 6 and 9 h) all resulted in cell division and release of motile cells when the material was resuspended in the original ‘aged’ medium, the length of immersion in the enriched medium having a direct influence on the time for which cells would continue dividing. Darkening of chromatophore colour, with apparent increase in carotenoid content, was a characteristic feature of the ‘recovery’ process. Extraction of pigments and measurement of absorption maxima showed that the same pigment content could be detected qualitatively both in the bleached ‘aged’ benthic cells and the ‘young’ benthic cells in fresh media. An unusual feature was observed in the changed staining properties of the leucosin reserve of the ‘aged’ benthic cells when treated with Cresyl Blue. Relatively small numbers of cells (ca 10–12% of a population) showed the characteristic rose-red colouration. In enriched media this proportion increased to ca 80 % over 3–4 days with increased light and temperature. Under similar conditions, aged cells in the ‘aged’ medium showed an increase in the cells giving a vesicle colouration over the first few days, but thereafter the numbers of cells so reacting steadily decreased. The results of these and other experiments suggested that the colour reaction is associated with certain levels of metabolic activity in the cells and there was no evidence that failure of the stain to penetrate the cell membrane was a cause of the lack of vesicle colour in a large proportion of the aged cells. The relevance of this experimental work to field observations on the benthic phases of Haptophyceae is also discussed.     

Prenatal diagnosis of Hurler's syndrome—Biochemical studies on the affected fetus

Summary A prenatal diagnosis of Hurler's syndrome was made in a pregnancy at risk in a family with two affected children. The fetus was diagnosed as having Hurler's syndrome on the basis of a deficiency of -L-iduronidase in the cultured amniotic cells. The glycosaminoglycans (GAG) content in the supernatant of the amniotic fluid was increased about 1.5 fold compared with that in the control, and increases of heparan sulfate and dermatan sulfate were observed on electrophoresis.The diagnosis could be confirmed by the deficiencies of -L-iduronidase in the liver and brain from the affected fetus. GAG content in the liver from the affected fetus was increased approximately 10 fold as compared with that in the control fetal liver, and most of the GAG were degraded. The GAG content was observed to be increased two fold in the brain, and dermatan sulfate, which was not detected in normal fetal brain, was identified. -galactosidase activities in the affected liver and brain were decreased to 30tt50% of the control, and an altered hexosaminidase A was also observed in the liver.     

Duplication of the hemoglobin α-chain gene in sheep: Characterization of a new α-chain variant present in animals possessing the αLeu and theIIαHis chains

The structural characterization of a third type of α chain, detected in the hemoglobin of sheep also possessing the α^Leu and the^IIα^His chains, is reported. The new α-chain variant is an allele of the common α^Leu chain controlled by the¹α locus and differs from it in the replacement of the serine residue at position 8 with an alanine (^Iα^{8 Ser→Ala}). The alanine variant was observed in 38 of 206 sheep, whose hemolysates were analyzed by isoelectric focusing, and was found exclusively among the 57 animals also possessing the^IIα^His chain. This research was supported in part by funds from the Italian CNR (Grant 01859.04) and from the Ministero Pubblica Istruzione.