Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Complete heavy and light chain variable region sequence of anti-arsonate monoclonal antibodies from BALB/c and A/J mice sharing the 36-60 idiotype are highly homologous

Structural and serologic studies on murine A/J monoclonal anti-arsonate antibodies resulted in the identification of a second idiotype family (Id36-60) in addition to the predominant idiotype family (IdCR). Id36-60, unlike IdCR, is a dominant idiotype in the BALB/c strain but is a "minor" idiotype in the A/J strain. The complete heavy and light chain variable region (VH and VL) amino acid sequences of a representative Id36-60 hybridoma protein from both the A/J and BALB/c strains have been determined. There are only four amino acid sequence differences between the VH of antibody 36-60 (A/J) and antibody 1210.7 (BALB/c). Two of these differences arise from single nucleotide changes in which the A/J and BALB/c Id36-60 VH germline gene sequences differ. The two other differences are the result of somatic mutation in hybridoma protein 36-60. In addition, Id36-60 heavy chains employ the same D and JH3 segments in both strains. The entire Vk2 VL of 36-60 and 1210.7 differ by only two amino acids, suggesting that like the heavy chains, they are derived from highly homologous VL genes. The same Jk segment is used in both antibodies. A comparison of the amino acid sequence data from Id36-60-bearing hybridomas suggests that a heavy chain amino acid difference accounts for the diminished arsonate binding by the 1210.7 hybridoma protein. Because the 1210.7 heavy chain is the unmutated product of the BALB/c VH gene, somatic mutation in VH may be required to enhance Ars affinity in this system.     

Molecular evidence for Acanthocephala as a subtaxon of Rotifera

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters. Correspondence to: J.R. Garey     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.     

Dopamine Receptors in Subcellular Fractions from Bovine Caudate: Enrichment of [3H]Spiperone Binding in a Postsynaptic Membrane Fraction

Abstract: Specific binding of tritiated dopamine, spiperone, and N-propylnorapomorphine was examined in subcellular fractions from bovine caudate nucleus. All fractions contained at least two sets of specific binding sites for [³H]spiperone (K_{D 1}^aPP= 0.2 nM, K_{D 2}^aPP= 2.2 nM), the higher affinity sites accounting for one-third to one-eighth of the total. [³H]Spiperone binding was slightly enriched over the total particulate fraction in P₂, P₃, SPM, and a crude fraction of synaptic mitochondria. A microsomal subfraction (P₃B₂) exhibited the highest specific binding capacity obtained, representing a fourfold enrichment over the total particulate fraction. [³H]Dopamine exhibited apparent binding to a single class of high-affinity sites in all fractions examined (K_D^aPP= 4.0 nM). A greater than twofold enrichment was observed in all fractions except myelin and P₃, with a fivefold enrichment in SPM and P₃B₂. At least two classes of receptors were labeled by [³H]-N-propylnorapomorphine (K_{D 1}^aPP= 0.55 nM, K_{D 2}^aPP= 20 nM), using 50 nM-spiperone together with 100 nM-dopamine to define nonspecific binding. Although binding to the higher affinity site was displaced by spiperone, and lower affinity binding by dopamine, comparison of receptor densities with values obtained by using [³H]spiperone and [³H]dopamine directly suggested that [³H]-N-propylnorapomorphine labeled additional sites. We have also examined a postsynaptic membrane (PSM) fraction obtained from SPM by successive extraction with salt and EGTA followed by sonication and separation on a density gradient. [³H]Spiperone binding in PSM was enriched two- to threefold over unfractionated SPM with a concomitant decrease in [³H]dopamine binding. The enrichment in spiperone receptors was almost entirely due to an increase in the number of lower affinity binding sites, suggesting that these sites may be associated with the postsynaptic membrane.     

Immunoglobulin mu- and gamma-ribonucleic acid sequences in thymocytes and splenocytes from normal and hyperimmune mice

The immunoglobulin heavy-chain ribonucleic acid (RNA) repertoire of mouse thymocytes was examined. Previously, this laboratory reported immunoglobulin alpha-chain RNA sequences in mouse thymocytes [Near, R. I., & Storb, U. (1979 Biochemistry, 18, 964]. We have extended these studies to encompass mu, gamma 2b, and gamma 1 heavy-chain RNA sequences, mu-, gamma 2b-, and gamma 1-messenger RNAs (mRNAs) were purified from myelomas to 45, 22, and 54% purity, respectively. Each of these mRNAs faithfully translated into the appropriate immunoprecipitable protein in a reticulocyte lysate translation system. The gamma 1-mRNA translated into two major immunoprecipitable products of about 52 500 and 51 000 daltons while mu- and gamma 2b-mRNAs yielded only a single major protein. Complementary deoxyribonucleic acids (cDNAs) prepared from the mRNAs were used as hybridization probes and revealed the presence of about 70 mu-RNA sequences per average thymocyte as determined by hybridization kinetics, while gamma 1 and gamma 2b sequences were at the limits of detection. The mu-RNA sequences are present in the cytoplasm and are greater than 50% polyadenylated. Upon hyperimmunization of mice with sheep red blood cells, gamma 1-RNA in splenocytes increased by about 100-fold while only slightly increasing in thymocytes. mu and gamma 2b increased 2-3-fold in splenocytes and only slightly in thymocytes. The results argue against RNA sequences appearing in thymocytes due to contamination with peripheral confirmed with cloned cDNA probes. Thymocyte RNA analyzed by Northern blots displayed bands of the same size as those in splenocyte RNA or in purified mRNA when hybridized to mu, gamma 2b and alpha cloned probes. Also, K light-chain RNAs of the same size were found in spleen and thymus by using a cloned K-DNA probe. The results are consistent with the thymus containing mu-, alpha-, and K- and small amounts of gamma 1- or gamma 2b-RNAs coding for heavy- and light-chain-like proteins which may play a role in T-cell function.     

Photoaffinity labeling of three renal cyclic 3',5'-adenosine monophosphate-binding proteins.

Evidence is presented for the presence of multiple cyclic AMP binding components in the plasma membrane and cytosol fractions of porcine renal cortex and medulla. N6-(Ethyl-2-diazomalonyl)-3',5'-adenosine monophosphate, a photoaffinity label for cyclic AMP binding sites, exhibits non-covalent binding characteristics similar to cyclic AMP in membrane and soluble fractions. Binding data for either compound to the plasma membrane fraction yields biphasic Scatchard plots while triphasic plots are obtained with the dialyzed cytosol. When covalently labeled fractions are separated on SDS-polyacrylamide gel electrophoresis, the cyclic AMP photoaffinity label is found on 49 000 and 130 000 dalton components in each kidney fraction. DEAE-cellulose and gel filtration chromatography of the labeled cortical cytosol fraction establishes that the three components suggested by the binding data correspond to two 49 000 dalton species and a 130 000 component. The 49 000 species have higher affinities for cyclic AMP than the 130 000 component (Ka(1) = 2.0 . 10(9), Ka(2) = 1.7 . 10(8), Ka(3) = 1.0 . 10(7)). The 49 000 components are associated with protein kinase activity while the 130 000 component does not exhibit protein kinase, adenosine deaminase, or cyclic nucleotide phosphodiesterase activity. Immunologic results and effects of phosphorylation and cyclic GMP on cyclic AMP binding further suggest that the 49 000 components are regulatory subunits of cyclic AMP-dependent protein kinases. Cyclic AMP binding to the 130 000 component is markedly inhibited by adenosine and adenine nucleotides, but not cyclic GMP. Thus, this component may reflect an aspect of adenosine control or metabolism which may or may not be a cyclic AMP-related cellular function.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies