An hydroxylapatite batch assay for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor complexes.

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D₃-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, $\text{v}\text{v}$) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D₃ receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D₃ and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.     

Response of human and chick kidney adenylate cyclase to biologically active synthetic fragments of human parathyroid hormone

The 34-amino acid NH₂-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall $\text{et al.}$ (1) is approximately 140 times more potent than the fragment synthesized according to Brewer $\text{et al}$ (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation $\text{67}\text{342}$ in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both $\text{in vivo}$ and $\text{in vitro}$.     

Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae

Mutations which inactivate the NADP-glutamate dehydrogenase (anabolic GDHase) pleiotropically release the ammonia inhibition (NH₄⁺ effect) on a number of distinct catabolic activities. In addition to releasing inhibition on several permeability functions (1), these mutations suppress the NH₄⁺ effect on the synthesis of arginase, urea amidolyase and allantoinase. They do not affect the NH₄⁺ effect on the NAD-glutamate dehydrogenase.Two mechanisms of action of these mutations have to be considered, namely a modification of the process of $\text{induction}$ (such as removal of inducer exclusion) and a suppression of $\text{nitrogen catabolite repression}$.     

Letter: Studies on histidinol dehydrogenase preliminary crystallographic data

Crystals of histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium have been obtained in large size suitable for single crystal X-ray studies. The following crystal data were obtained on examination of a number of X-ray precession photographs; crystal system monoclinic, $\text{a = 149.6}\text{A}\text{?}$, $\text{b = 88.9}\text{A}\text{?}$, $\text{c = 105.6}\text{A}\text{?}$, β = 124.5 °, space group C2. The density of the crystal measured by flotation in bromobenzene/xylene mixture is 1.186 g/cm³. There is one dimer molecule of molecular weight 80,000 in one crystallographically asymmetric unit.     

Proton nuclear magnetic resonance studies of the manganese (II) binding site of concanavalin A. Effect of saccharides on the solvent relaxation rates

The longitudinal relaxation rate ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$) of water protons was studied in solutions of Mn(II)-concanavalin A at a number of frequencies. These relaxation rates were lowered in the presence of a variety of saccharides which have affinities for concanavalin A which range over two orders of magnitude. A good correlation was found in which saccharides which bind tightly have the greatest effect and saccharides which bind weakly or not at all have little effect on the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ values. The temperature dependence of the proton relaxation rates showed that the lowering of these rates in the presence of saccharides was most likely due to a change in the exchange rate of solvent interacting with protein-bound Mn(II), $\text{1}\text{T}{}_{\mathrm{<mtext>m</mtext>}}$.An analysis of the temperature and frequency dependence of the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ (transverse) solvent proton relaxation rates resulted in evaluation of a number of parameters for solvent water molecules interacting in the first coordination sphere of Mn(II) bound to concanavalin A. The ratio of the number of water molecules (q) to the Mn(II)-proton distance (r) obtained from a computer fit of the data over a limited temperature range is in accord with the findings of Koenig et al. ((1973) Proc. Nat. Acad. Sci.70, 475) and Meirovitch and Kalb ((1973) Biochim. Biophys. Acta303, 258). However, our studies of $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ of water over a more extensive temperature range are best fit with the following conclusions: at low temperatures (<20 °C), the data are consistent with an outer-sphere relaxation process. At higher temperatures (> 30 °C), the water molecule in the inner coordination sphere of the bound Mn(II) begins exchanging more rapidly and contributes to the relaxation processes ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$). The relaxation time of protons in the inner coordination shell, T_1M, contributes over the entire temperature range and produces a frequency dependence in the relaxivity data from 6 to 100 MH_z since the contributions to the correlation times are in the range 10^?9-10^?8 sec.     

Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Isopycnic-zonal centrifugation of plasma membrane,sarcoplasmic reticular fragments,lysosomes, and cytoplasmic proteins from phasic skeletal muscle

Homogenates of the posterior latissimus dorsi muscle, a phasic muscle, were fractionated by a one-step zonal centrifugation technique into four major organelle populations and cytoplasmic constituents. These were: (1) Plasma membrane fragments with a modal equilibrium density of 1.10 and containing 5′-nucleotidase, alkaline phosphodiesterase, $\text{p-}\text{nitrophenylphosphatase}$ and acid phosphatase (β-glycerophosphate was used as the substrate). (2) Sarcoplasmic reticular fragments which could be further subdivided into calcium transport vesicles, with a modal equilibrium density of 1.16, that exhibited calcium uptake; K⁺-ATPase; leucyl-β-naphthylamidase; acid phosphodiesterase; acid phosphatase (using cytidine monophosphate as the substrate); and sarcoplasmic reticular lysosomes, with a modal equilibrium density of 1.18, possessing dipeptidyl-aminopeptidase II, cathepsin D, α-glucosidase, $\text{N-}\text{acetyl}\text{-β-}\text{glucosaminidase}$, and NADH oxidase activity. (3) Mitochondria with a modal equilibrium density of 1.21. (4) Catalase-containing vesicles with a modal equilibrium density of 1.22; and cytoplasmic constituents (modal density of 1.25) with phosphorylase, pyruvate kinase, myosin-ATPase, aldolase, and protein and RNA content. The purity of these organelles was equal to or better than previous efforts, with a 30-fold purification achieved for 5′-nucleotidase and alkaline phosphodiesterase. These results lend support to the hypothesis that the sarcoplasmic reticulum of phasic muscle, in addition to its specialized role in excitation-contraction coupling, represents a multifunctional membrane system, and that, similar to the smooth endoplasmic reticulum of other cells, it includes some membrane-bound lysosomal enzymes and NADH oxidase.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules ($\text{S}{}_{\mathrm{<mtext>H</mtext>}}\text{(}\text{average sedimentation coefficient}\text{) = 12 400}\text{S}$ in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin ($\text{?95\%}$ of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium.The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

Insect yolk protein precursor, a juvenile hormone induced phosphoprotein

The juvenile hormone induced vitellogenic female-specific protein of $\text{Leucophaea}$$\text{maderae}$ was isolated from hemolymph of egg maturing females on DEAE or QAE anion-exchange columns. Minor contaminants could be removed by centrifugation to equilibrium on CsCl gradients. The buoyant density of this purified protein is 1.344 g/ml. It is a lipophosphoprotein of low phosphorus (0.14%) content. Essentially all ³²P label from in vivo labelled protein was recovered in phosphoserine. The amino acid residues of the vitellogenic protein compare well with the purified yolk protein.     

Evaluation of the mixed interaction between apolipoproteins A-II and C-I by equilibrium sedimentation

The mixed interaction between human apolipoproteins C-I and A-II, each of which self-associate in aqueous solution, has been evaluated by sedimentation equilibrium measurements. In order to simplify data analysis apoC-I and apoA-II were modified by treatment with 2-nitrophenylsulfenyl chloride and tetranitromethane respectively. The molecular properties of the resulting derivatives, S-apoC-I and N-apoA-II, each of which have appreciable extinction coefficients above 350 run, were indistinguishable from the corresponding unmodified species. Sedimentation equilibrium data were obtained with mixtures of S-apoC-I and native apoA-II, N-apoA-II and native apoC-I, and native apoC-I and native apoA-II. Mixed complex formation was detected readily with all mixtures investigated. The combined results were most consistent with a single mixed oligomer containing 2 molecules of apoA-II and 4 molecules of apoC-I. The corresponding equilibrium constant was 31 248 ± 890 ($\text{XXX}\text{gm}$)⁵.     

Dansylation of bacteriorhodopsin near the retinal attachment site

The purple membrane of $\text{Halobacterium}\text{halobium}$ was reacted with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) at pH 8.0. Chromophoric and functional properties of the product appear unaltered. Approximately 2 moles of dansyl group were incorporated per mole of bacteriorhodopsin, part bound to bacteriorhodopsin and part bound to lipids. Purification and fragmentation of the protein showed most of the dansyl modification in a fragment containing residues 33 to 56. Amino acid analysis indicates that the major dansylated site is lysine 40. We conclude that, contrary to published models, 1) bacteriorhodopsin folds in a way that exposes lysine 40 at the membrane surface, and 2) this side chain is not involved in the proton pump mechanism.     

1Alpha,25-dihydroxyvitamin D3 receptor: competitive binding of vitamin D analogs

The intestinal nuclear receptor for lα,25-dihydroxyvitamin D₃ has been utilized to determine the ability of vitamin D-active sterols to compete with this hormone at the molecular level. 25-Hydroxyvitamin D₃ and lα-hydroxyvitamin D₃ must be present in 150 and 450 times the concentration respectively of lα,25-dihydroxyvitamin D₃, $\text{in}$$\text{vitro}$, to displace the physiologic hormone. These data indicate that: i) superphysiologic levels of 25-hydroxyvitamin D₃ may simulate lα,25-dihydroxyvitamin D₃ and act directly on isolated target organs and ii) the biologic potency observed for low doses of lα-hydroxyvitamin D₃, $\text{in}$$\text{vivo}$, is probably the result of 25-hidroxylation of the lα-derivative to form lα,25-dihydroxyvitamin D₃.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

Equilibrium centrifugation of proteins in acidic solutions. Beta-lactoglobulin A in aqueous acetic, propionic, and butyric acids.

Short chain aliphatic acids are almost neutrally buoyant in aqueous solutions, and preferential interaction of macromolecules with these solvent components should not greatly affect apparent molecular weights determined by equilibrium ultracentrifugation. The feasibility of molecular weight estimations using native, neutral pH values of partial specific volume has been tested: equilibrium ultracentrifugation of β-lactoglobulin A (β-LgA) has been carried out in aqueous acetic, propionic, and butyric acids in the absence of any other added electrolyte. These solutions are highly nonideal because of the extreme Donnan effect. Apparent molecular weights estimated at infinite dilution using the native neutral pH value of the partial specific volume, $\text{v}{}_{\mathrm{p}}$, differed by less than 5% from the monomer formula weight. The 10 m acids appear to be least effective as dissociating agents for β-LgA, with a weak reversible monomer-dimer association suggested in 10 m acetic acid, with significant heterogeneity apparent in 10 m propionic acid, and with a lack of direct solubility in 10 m butyric acid. All the 0.1 m acids and all the 1 m acids were essentially equally effective as dissociating agents, with the exception of 1 m butyric acid which dissolved β-LgA only slowly to give significantly heterogeneous solutions. From these results and from our previous experiments with aldolase (6), it appears feasible to use the native values of $\text{v}{}_{\mathrm{p}}$ to obtain estimates of molecular weights of proteins in aqueous organic acids as dissociating agents.     

Synthesis of α-amylase and α-glucosidase by membrane bound ribosomes from Bacilluslicheniformis

α-Glucosidase was membrane bound during exponential growth of $\text{Bacillus}\text{licheniformis}$ but was released into the medium during stationary phase. It could be partially removed from exponential phase cells by washing with NaCl (0.5 M). α-Amylase was exclusively extracellular and could not be detected in cells. Polysomes were prepared from exponential phase cells and separated into membrane bound and soluble fractions. $\text{In}\text{vitro}$ chain completion and immunoprecipitation showed that α-glucosidase and α-amylase were synthesized by membrane bound and not by soluble ribosomes.     

Bile acid biotransformation rates of selected gram-positive and gram-negative intestinal anaerobic bacteria.

Treatment of whole cell suspensions of $\text{Eubacterium aerofaciens}$ and $\text{Bacteroides fragilis}$ with lysozyme resulted in a marked increase (>100-fold) in the rates of biotransformation of cholate to 7-ketodeoxycholate (7-KD) in the former but only a 2-fold increase in the latter bacterium. In $\text{B. fragilis}$ the total activity of both NAD-dependent 7-α-hydroxysteroid dehydrogenase (7-α-OHSDH) and bile salt hydrolase (BSH) increase markedly during the stationary growth phase. Both enzymes were found in the spheroplast lysate and the Triton-soluble washed membrane fractions but only BSH was found in the spheroplast medium.     

Association of α2-macroglobulin-thrombin and α2-macroglobulin-plasmin complexes with isolated hepatocytes

¹²⁵I-labelled $\text{α}{}_2\text{-}\text{macroglobulin}$ complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values calculated from displacement experiments were 7.9 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ and 8.5 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. $\text{α}{}_2\text{-}\text{Macroglobulin}$ itself bound also to hepatocytes, but the affinity of the $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes was higher than that of the inhibitor alone, and $\text{α}{}_2\text{-}\text{macroglobulin}$ was not internalized, either. ¹²⁵I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes. The $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ complex competed with the $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$ complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for $\text{α}{}_2\text{-}\text{macroglobulin-proteinase}$ and antithrombin III-proteinase complexes are different.     

Specificity reversal in phospholipase A2 hydrolysis of lipid mixtures

The activity and specificity of phospholipase A₂ from cobra venom ($\text{Naja naja naja}$) toward binary mixtures of phosphatidylcholine and phosphatidylethanolamine in mixed micelles with the nonionic surfactant Triton X-100 were examined. In mixtures containing 5–50 mol % phosphatidylcholine, the rate for phosphatidylethanolamine hydrolysis was enhanced greatly over that for phosphatidylcholine. This is in marked contrast to previous studies with individual phospholipid species in mixed micelles where phosphatidylcholine was found to be the preferred substrate and phosphatidylethanolamine was found to be a very poor substrate. Possible explanations for this specificity reversal are considered.     

Labeling of bovine corpus luteal plasma membrane human chorionic gonadotropin or luteinizing hormone (hCG/LH) receptor and its purification and properties.

A method has been developed for labeling receptors for human chorionic gonadotropin or luteinizing hormone ($\text{hCG}\text{LH}$) present on bovine corpus luteal plasma membranes. It consists of four steps: (a) protection of the receptor by treating the plasma membranes with hCG; (b) iodination of the membranes with KI using glucose, glucose oxidase, and lactoperoxidase; (c) unmasking the receptor with either 2 m NaCl, 1 m guanidine hydrochloride, or rabbit anti-hCG; and (d) reiodination of the membranes using Na¹³¹I. After solubilization by successive treatments with Sepharose-concanavalin A and Sepharose-hCG and finally by preparative disc electrophoresis, the resulting purified receptor after electrophoresis in polyacrylamide gel showed a single radioactive band containing receptor activity. This highly purified receptor is fairly stable and retains its hormonal specificity, binding affinity, and pH optimum. It was observed that the receptor alone or as a complex with the hormone tends to aggregate. The receptorhormone complex does not dissociate during polyacrylamide-gel electrophoresis.