Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10⁶-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp. Physiol. Ser. B130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physiol. Biochem.87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. (Arch. Biochem. Biophys.196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.     

Characterization and comparison of the major glycoprotein from three strains of Rous sarcoma virus.

The major glycoprotein g2 was purified from three strains of Rous sarcoma virus, representing subgroups A, B, and C. Carbohydrate analysis showed that glucosamine, mannose, galactose, fucose and sialic acid constitute 40% of the weight of the subgroup A glycoprotein and 15% of the subgroup B and C glycoproteins. The molar ratios of sugars were very similar and amino acid compositions were similar but not identical for the three glycoproteins. Glycosidase digestions of subgroup A and C glycoproteins suggested the presence of two types of oligosaccharide chains, the complex serum type, with terminal sequences sialic acidα-Galβ-GlcNAcβ- and the high mannose type with terminal α-linked mannosyl residues. After removal of 70% of the carbohydrate by glycosidases, subgroup A glycoprotein contained only glucosamine and mannose, in the molar ratio 2.0:1.3. The sequence of sugar release was consistent with oligosaccharide structures such as those which have been described for other glycoproteins. The plant lectins concanavalin A and wheat germ agglutinin were shown to interact strongly with the g2 glycoprotein from viruses of all three subgroups.     

Purification of microtubule protein from beef brain and comparison of the assembly requirements for neuronal microtubules isolated from beef and hog.

The polymerization of microtubule protein from beef brain is inefficient under the same conditions which are optimal for the assembly of microtubules isolated from hog brain (0.1 m piperazine-N,N′-bis(2-ethanesulfonic acid) buffer at pH 6.94). In examining the conditions required for microtubule polymerization in both beef brain extract and purified microtuble protein, it was determined that the pH optimum was pH 6.62 or 0.3 pH unit lower than the reported optimum for hog. Other assembly requirements (ionic strength, Mg²⁺ and nucleotide concentration, temperature) remained essentially the same as for hog. By separating and recombining fractions of tubulin and nontubulin components prepared from beef and hog microtubule protein, the requirement for the reduction in pH was found to be due to the tubulin and not to the microtubule-associated proteins. It was also determined that the efficiency of beef tubulin assembly, as measured by the yield of microtubule polymer, decreased rapidly after slaughter with a half-time of 19 min. Furthermore, when the overall efficiency of polymerization was reduced, the extent of assembly at each cycle of purification by disassembly and assembly was also observed to be depressed. The variations in the requirements for neuronal tubulin assembly in two closely related mammals suggest that the conditions required for assembly of microtubule protein in other tissues and cell types may also be different.     

α-Bungarotoxin binding in the central nervous system of Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin in the central nervous system of the horseshoe crab, Limulus polyphemus, was investigated. Comparative binding studies in various tissues of L. polyphemus demonstrated a selective association of the toxin with nervous tissues. The greatest enrichment of toxin binding in subcellular fractions of brain tissue was observed in a fraction enriched in mitochondria and acetylcholinesterase-containing membranes. Autoradiographic studies revealed the localization of α-bungarotoxin binding to the longitudinal connectives and neuropile regions of the abdominal ganglia. Three toxin binding components with approximate sedimentation coefficients of 9 S, 15.4 S and 17.4 S were present in solubilized extracts of brain tissue. ¹²⁵I-labeled α-bungarotoxin binding to these components was inhibited 72%, 47%, 9% and 0% by 10 μM concentrations of (+)-tubocurarine, nicotine, scopolamine and pilocarpine, respectively. The apparent formation of the 15.4 S and 17.4 S proteins from the 9 S protein was obtained. The 15.4 S and 17.4 S components are suggested as aggregates of the 9 S protein. This 9 S protein is proposed as an acetylcholine receptor from the central nervous system of L. polyphemus.     

Studies on spectral characteristics of allophycocyanin isolated from Anabaena cylindrical: Curve-fitting analysis

Spectroscopic characteristics of allophycocyanin (APC) isolated from Anabaena cylindrica were studied by curve-fitting analyses of absorption spectra measured at room temperature. Gaussian curve-fittings based on the least-squares method were done on absorption spectra for three different dissociation-association conditions of the protein. The composition of the component bands was similar in the three absorption spectra and could be classified into three groups depending on changes in band intensity; the first group was represented by the main band at 653 nm, which was intense in the trimer APC but markedly reduced in the monomer. The second group was represented by the main band at 607 nm, which showed opposite changes. The third group had its main band at 623 nm, and it remained the same in the three absorption spectra. The sum of the band area of the variable components (the first and second groups) was almost equal to that of the constant components (the third group). These results indicate that: (i) APC has two types of phycocyanobilin (PCB) which differ in their spectroscopic characteristics. (ii) The PCB responsible for the variable component bands, and thus for the spectral changes, has two excitation states corresponding to 653 and 607 nm, respectively, and the spectral changes are ascribable to variations in the relative frequencies of occurrence of the two states: in associated APC the first state predominates, and in dissociated APC, the second. (iii) PCB for the constant component bands has only one excitation state corresponding to the 623-nm band. Changes in the emission spectra correlate with these changes in the absorption spectra, suggesting that in APC the phycocyanobilins responsible for and independent of absorption spectrum changes act as the fluorescing and sensitizing types, respectively.     

The role of copper and quaternary structure on the conformational properties of Octopus vulgaris hemocyanin

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Retention of Ca2+ by rat liver and rat heart mitochondria: Effect of phosphate,Mg2+, and NAD(P) redox state

Parallel measurements of Ca²⁺ uptake, oxygen consumption, endogenous Mg²⁺ efflux, and swelling in rotenone-poisoned rat liver and rat heart mitochondria showed that heart mitochondria is much more resistant to uncoupling by Ca²⁺ in the presence of phosphate than rat liver mitochondria. The extent of Mg²⁺ efflux and swelling induced by Ca²⁺ accumulation are much less pronounced in heart mitochondria. Uncoupling and swelling in liver mitochondria seem to result from the loss of membrane-bound Mg²⁺ as a consequence of Ca²⁺ recycling across the membrane as induced by phosphate. Exogenous Mg²⁺ protects liver mitochondria against the deleterious effects of Ca²⁺ by inhibiting a ruthenium red-insensitive Ca²⁺ efflux induced by phosphate. Phosphate does not induce recycling of Ca²⁺ in heart mitochondria. On the other hand, heart mitochondria respiring on NAD-linked substrates or with succinate in the absence of rotenone behave like liver mitochondria with respect to the alterations caused by Ca²⁺ recycling. In heart mitochondria the recycling of Ca²⁺ is related to the redox state of pyridine nucleotides, which suggests that the ruthenium red-insensitive efflux of Ca²⁺ is subject to metabolic control. In addition it has been observed that Sr²⁺does not undergo cyclic movements across the membrane. The data indicate that the efflux pathway is more specific for Ca²⁺ than the ruthenium red-sensitive influx transporter.     

Decomposition of BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) in aqueous solution

BCNU, labelled with ¹⁴C in the chloroethyl groups, decomposes in neutral aqueous solution to release half of its radioactivity as volatile products. These have been identified by a combination of gas chromatography and mass spectrometry as vinyl chloride, acetaldehyde, dichloroethane, and chloroethanol. This set of products is consistent with the existence of chloroethylcarbonium ions as reactive intermediates which would produce the previously described substituted nucleotides.     

Structural investigations on the lipopolysaccharide isolated from Vibrio cholera,Inaba 569 B

On hydrolysis, the purified lipopolysaccharide (LPS) isolated from Vibrio cholera, Inaba 569 B, yielded glucose, mannose, a heptose behaving like d-glycero-l-manno-heptose and one behaving like d-glycero-l-gluco-heptose, 2-amino-2-deoxy-glucose, and glucuronic acid in the molar ratios of ～9:4:5:1:2:5. Studies on the LPS, the polysaccharide (PS), and carboxyl-reduced LPS showed that the PS has a branched structure, with (1→2)-linked mannopyranosyl and a heptopyranosyl, and (1→4)-linked glucopyranosyluronic and 2-amino-2-deoxyglucopyranosyl residues in the interior part of the molecule, and glucopyranosyl and heptopyranosyl residues as nonreducing end-groups.     

Structural studies on a carbohydrate chain isolated from the lipopolysaccharide of Shigella boydii type 8

The lipopolysaccharide isolated from the cells of Shigella boydii type 8 bacteria gave precipitin bands against homologous antisera on Ouchterlony plates, whereas the carbohydrate-containing fractions obtained from it did not. One of the fractions was obtained in major proportion and contained 23.5% of sugars. A structure was assigned to the carbohydrate chain in this material by using the results of methylation, periodate oxidation, and deamination studies.     

Semiquantitative paper chromatographic separation of chlorophylls a and b

[¹⁴C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chl b (as pheophytin b) may be reduced to 0.15–0.35.     

Structural and functional studies of ribonucleoprotein fragments isolated from Escherichia coli 50 S ribosomal subunits.

Ribonuclease digestion of 50 S-derived LiCl cores led to 22 ribonucleoprotein particles which were isolated by repeated sucrose gradient centrifugations. The protein content was determined and ranged from 2 to 28 proteins. Most of the fragments showed a unique RNA pattern as judged by acrylamide gel electrophoresis.Functional tests were performed with selected fragments. No fragment was active in the poly(U) or the peptidyl-transferase assay. Chloramphenicol binding studies revealed that in addition to the dominant role of protein L16, the protein L11 (or L6) is involved directly in the drug binding. Finally, tests for ATPase and GTPase activity showed that protein L18 is involved in GTPase activity.     

In Vivo and in Vitro binding of platinum to metallothionein

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammine-platinum(II)). Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl₂ significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl₂ pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.     

Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.     

Application of CNDO2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

$\text{CNDO}\text{2}$ molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond distance = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond distance = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

In vivo and in vitro incorporation of endogenous nucleotides by the energy-transducing ATPase of Streptococcus faecalis

The soluble ATPase isolated from Streptococcus faecalis membranes containing tightly bound endogenous nucleotides do not exchange in the presence of ATP and Mg+2 added during the purification of the enzyme. In this paper the stoichiometry of endogenous nucleotides in the soluble ATPase obtained from (a) growing cells, (b) nongrowing glycolyzing cells, and (c) isolated cell membranes has been defined. The time course of incorporation was also studied in nongrowing, glycolyzing cells and isolated cell membranes. In all cases, 1-2 mol of nucleotide was bound per mol of enzyme. Maximal incorporation required approximately 1 h at 38 degrees C. Incorporation of cytoplasmic nucleotide into the enzyme occurred by a process of slow exchange for bound nucleotide. N,N'-dicyclohexylcarbodiimide, which inhibits the membrane-bound ATPase and prevents generation of the protonmotive force, had no effect on incorporation of endogenous nucleotides in glycolyzing cells. Treatment of glycolyzing cells with gramicidin D plus K+, which dissipates the protonmotive force but has no effect on ATPase activity, did not inhibit incorporation of nucleotide. These results support the view that the slow exchange-incorporation of endogenous nucleotide(s) is independent of ATP hydrolysis and a protonmotive force. An in vitro system for the study of nucleotide binding at endogenous sites is described.     

Synthesis and characterization of lipid-linked mannosyl oligosaccharides in Volvox carteri f. nagariensis

Particulate membrane fractions from Volvox carteri catalyze the transfer of mannose from GDP-mannose to dolichyl diphosphate-[¹⁴C]chitobiose to form lipid-linked oligosaccharides up to a dolichyl diphospnate-chitobiose-(mannose)₅ structure. Mannosylation of the chitobiosyl lipid requires divalent cations and detergents as solubilizing agents. Depending on the nature of the detergent, the oligosaccharide pattern differs markedly: With deoxycholate or the zwitterionic detergent 314 a lipid-linked trisaccharide accumulates. The nonionic Triton X-100, however, gives rise to a spectrum of compounds up to a heptasaccharide. Enzyme digestion of the tri- and pentasaccharide structure, obtained after mild acid hydrolysis of the corresponding [¹⁴C]glycolipids, revealed that the first mannose is bound via a β-glycosidic linkage to the chitobiosyl core, whereas the outer mannose residues are linked as α-mannosides. Our studies indicate that, in agreement with recent findings in other organisms, the innermost α-mannosidic residues are donated directly from GDP-mannose. The structure of oligosaccharides synthesized by Volvox membranes is thus consistent with results from other eucaryotic species, suggesting a common pathway of N-glycosylation of glycoproteins.     

In vitro synthesis of mycolic acids by the fluffy layer fraction of Bacterionema matruchotii

Biosynthetic activity for mycolic acid occurred in the fluffy layer fraction but not in the 5000g supernatant of Bacterionema matruchotii. With [1-¹⁴C]palmitic acid as precursor for the in vitro system, the predominant product was identified as C_32:0 mycolic acid by radio-gas-liquid chromatographie (radio-GLC) and gas chromatographic/mass spectroscopic analyses; if [1-¹⁴C]stearic acid was used, two major radioactive peaks appeared on GLC: one corresponding to the peak of (C_34:0 + C_34:1) mycolic acids and the other to (C_36:0 + C_36:1) mycolic acids. By pyrolysis/radio-GLC analysis, C_32:0 mycolic acid synthesized by [1-¹⁴C]palmitic acid was pyrolyzed at 300 °C to form palmitaldehyde (the mero moiety) and methyl palmitate (the branch moiety). The pH optimum for the incorporation of [1-¹⁴C]palmitate into bacterionema mycolic acids was 6.4 and the reaction required a divalent cation. The in vitro system utilized myristic, palmitic, stearic and oleic acids (probably via their activated forms) well as precursors, among which myristic and palmitic acids were more effective than the rest. Avidin showed no effect on the biosynthesis of mycolic acid from ¹⁴C-palmitate whereas cerulenin, a specific inhibitor of β-ketoacyl synthetase in de novo fatty acid synthesis, inhibited the reaction at a relatively higher concentration. Thin-layer chromatographic analysis of lipids extracted from the reacting mixture without alkaline hydrolysis showed that both exogenous [1-¹⁴] fatty acid and synthesized mycolic acids were bound to an unknown compound by an alkali-labile linkage and this association seemed to occur prior to the condensation of two molecules of fatty acid.