Peripheral Subunits Research Articles

Dissociation of the sodium-ion-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and reconstitution of the active complex from the isolated subunits.

Oxaloacetate decarboxylase was reconstituted from the purified alpha subunit and a Triton X-100 extract of bacterial membranes devoid of this protein. Upon freezing of oxaloacetate decarboxylase in salt solutions, the enzyme was split into subunits and the catalytic activity was abolished. The catalytically active decarboxylase complex was reconstituted by decreasing the salt concentration of the dissociated sample. The conditions for the inactivation were critical for an optimum recovery of catalytically active enzyme during reconstitution, and modest dissociating conditions generally improved the yield of the reconstitutively active decarboxylase. The dissociated enzyme has been separated by chromatography on avidin-Sepharose into two fractions: fraction I, that was not retained on the column, consisted of the beta + gamma subunits, and fraction II consisted of the biotin-containing alpha subunit. Oxaloacetate decarboxylase was reconstituted from a mixture of the isolated alpha and beta + gamma subunits. The Na+ transport activity was recovered, if a mixture of subunits alpha and beta + gamma was incorporated into liposomes, or by a sequential reconstitution, starting with the formation of proteoliposomes with the integral membrane proteins beta + gamma and completed by an attachment of the peripheral subunit alpha.

Multiple ubiquitination of vertebrate calmodulin by reticulocyte lysate and inhibition of calmodulin conjugation by phosphorylase kinase.

Mammalian calmodulin containing trimethyllysine 115 can be covalently coupled to ubiquitin in a Ca2+-dependent manner in the presence of ATP/Mg2+ by reticulocyte lysate. This conjugation reaction can be quantitated in a novel test employing fluphenazine-Sepharose. It is shown that at least 3 ubiquitin molecules can be coupled to calmodulin indicating that more than one lysine residue is involved in the ubiquitination reaction. In addition only the free form of calmodulin can be ubiquitinated. Neither calmodulin bound to phosphorylase kinase as an integral subunit (delta-subunit) nor that bound as a peripheral subunit (delta'-subunit) is ubiquitinated. A total binding of equimolar calmodulin to phosphorylase kinase occurs since the affinity of binding of calmodulin to phosphorylase kinase as integral (KCaMm unknown) or peripheral subunit (KCaMm ca. 30-50nM) is several order of magnitude higher than the corresponding affinity of calmodulin for the ubiquitin-conjugating enzyme (KCaMm ca. 8 microM). We conclude that the "protective" effect of phosphorylase kinase towards calmodulin conjugation is due to a changed conformation of bound calmodulin and/or inacessibility of the ubiquitination sites (e.g. at subunit-subunit interface). Thus Ca2+-dependent ubiquitination only of free calmodulin may provide an efficient scavanging mechanism (with subsequent breakdown) for all free calmodulin in excess of that amount which can be bound by the calmodulin-binding proteins in the cell.

Microtubular protofilaments and subunits of the outer dynein arm in cilia from dogs with primary ciliary dyskinesia.

Using a procedure of membrane solubilization followed by tannic acid fixation-staining, we found abnormally constructed central and B microtubules in cilia from dogs with primary ciliary dyskinesia. We also found that a ciliary abnormality commonly associated with primary ciliary dyskinesia, "shortened outer dynein arms," actually represents the absence of a specific set of the 3 peripheral subunits that make up the outer dynein arms. We believe that this is the first time that these have been reported. Because the proximal subunits of the outer dynein arm are present and attached to the A microtubule, we argue that in these patients there is no structural abnormality of the A microtubule or the A-tubule attachment sites for outer and inner arms and radial spokes. Also, because a majority of cilia in these patients were motile, we believe that the data from this study support the concept that the outer dynein arms are not required for some motion but probably required for normal motility; however, the central microtubules, central microtubular complex, and probably the B tubules are essential for motility.

The interaction of 1-anilino-8-naphthalene sulphonate with human Clq

Clq has 12 binding sites for l-anilino-8-naphthalene sulphonate (ANS), two per peripheral subunit. This number increases to 18 upon weak-acid-induced conformational transition in the globular heads. One ANS binding site is present in each C γ2 domain of human IgG. ANS is bound by Clq with a higher affinity ( K a = 2.07 × 10 6 M −1) than by the Fc fragment ( K a = 9.07 × 10 4 M −1) of human IgGl. Hence the inhibitory capacity of Clq binding to IgG immune complexes of ANS probably reflects its preferential binding to the globular heads of Clq. The characteristics of ANS-Clq binding may in part explain the hydrophobic component of the Clq-IgG interaction. It is suggested that an ionic-hydrophobic two-step process is involved in the contact between Clq and IgG.

In vitro synthesis and assembly of the peripheral subunits of coupling factor CF 1 (α and β) by thylakoid-bound ribosomes

Bispecific antisera were prepared to a mixture of thylakoid membrane polypeptides 4.1 and 4.2. The identity of these polypeptides as the α and β subunits of coupling factor 1 (CF 1) was established based on the cross-reactivity of the antisera toward CF 1 from peas and by an analysis of the thm-24 mutant of Chlamydomonas which lacks the CF 1 ATPase. Photochemical labeling of thylakoid membranes with hydrophobic and hydrophilic fluorescent probes indicated that these polypeptides did not significantly penetrate the membrane bilayer. Immunoprecipitation of the translation products of thylakoid-bound and soluble ribosomes showed the thylakoids to be the major site of synthesis of the polypeptides. Immunoprecipitation of the products of translation of total cellular RNA in a reticulocyte lysate showed no evidence for substantially higher molecular weight precursors. Further analysis of the thylakoid-bound synthesis of α and β revealed that some of the in vitro synthesized polypeptides had been incorporated into the CF 0CF 1 complex based on their release from membranes with trypsin and copurification with the CF 0-CF 1 ATPase.

Propionyl-coenzyme A carboxylase of Mycobacterium smegmatis. An electron microscopic study.

Propionyl-CoA carboxylase has been purified to homogeneity and examined in electron microscope. The native carboxylase presents a profile with a large central subunit to which smaller subunits are attached. The central subunit has two prominent profiles, one circular (100 A) with a central hole and the other rectangular (70 X 100 A). The six polypeptides of this subunit appear to be arranged in a cylindrical structure. Six spherical (50 A) biotin-containing peripheral subunits are attached in sets of three to the two opposite circular faces of the central subunit. A model of the 18-S carboxylase is presented.

Interaction between Schistosoma mansoni and the complement system: role of IgG Fc peptides in the activation of the classical pathway by schistosomula.

It was previously reported that Schistosoma mansoni schistosomula in the presence of IgG activate complement through the classical pathway (CCP). In the present work we have demonstrated that schistosomula incubated with IgG peptides resulting from IgG hydrolysis by schistosomula proteases are able to initiate complement activation through CCP, since a marked consumption of C1, C4, and C2 was observed. Our results eliminate an eventual direct action of the substances released by schistosomula on the activation of CCP. The first step of CCP activation required the preliminary binding of IgG peptides on the schistosomula surface. This interaction induced an increase in the C1q uptake by schistosomula. Since the involvement of the Fc portion of IgG molecules has been clearly evidenced, in this case the peripheral globular subunits of C1q recognize the CH2 region on IgG peptides or intact IgG molecules. By this mechanism, the local consumption of complement around schistosomula could contribute to its survival in the host.

Affinity partitioning of membranes. Evidence for discrete membrane domains containing cholinergic receptor.

Subsynaptic membrane domains from Torpedo californica electroplax contain nicotinic cholinergic receptor molecules at densities as high as 20,000 micrometers-2. Intense homogenization of the electroplax releases membrane fragments enriched in nicotinic receptor from basal lamina and other synaptic cleft and presynaptic elements. Ideally, preparations of membrane fragments, highly enriched in nicotinic receptor, should approach 125I-alpha-bungarotoxin-specific binding activities near the levels observed after receptor dispersal in detergents and subsequent affinity chromatography. We report the application of affinity partitioning, combined with multiple extraction techniques, to yield preparations of virtually homogeneous membranes enriched in nicotinic receptor alpha, beta, gamma, and delta subunits as well as the 43,000-dalton peripheral protein subunit. The countercurrent distribution technique serves to resolve three populations of receptor-containing membranes. One fraction is refractory to affinity partitioning and may represent aggregates of receptor-rich membranes with fragments derived from nonsynaptic membranes. The second and third fractions contain membrane fragments derived from the subsynaptic membrane and are highly enriched in nicotinic receptor (5.1 to 7.8 nmol of alpha-bungarotoxin binding sites/mg of protein). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of all three fractions indicates that alpha, beta, and gamma subunits are present in stable stoichiometric ratios, while the 43,000-dalton peripheral protein content varies by 33% between the fractions. However, removal of 90% of the 43,000-dalton component by mild alkali treatment does not result in conversion of one fraction into the other. The combination of affinity partitioning and counter-current distribution techniques utilized in this study should prove useful in the resolution of a variety of subcellular particles that contain specific binding sites.

Existence of multiple sites for ANS in an alpha-fetoprotein fraction demonstration by fluorescence polarization

Clq has 12 binding sites for l-anilino-8-naphthalene sulphonate (ANS), two per peripheral subunit. This number increases to 18 upon weak-acid-induced conformational transition in the globular heads. One ANS binding site is present in each Cγ2 domain of human IgG. ANS is bound by Clq with a higher affinity (Ka = 2.07 × 106M−1) than by the Fc fragment (Ka = 9.07 × 104M−1) of human IgGl. Hence the inhibitory capacity of Clq binding to IgG immune complexes of ANS probably reflects its preferential binding to the globular heads of Clq. The characteristics of ANS-Clq binding may in part explain the hydrophobic component of the Clq-IgG interaction. It is suggested that an ionic-hydrophobic two-step process is involved in the contact between Clq and IgG.

Reaction between the isolated globular sub-units of the complement component Clq and IgG-complexes

The globular sub-units of the Clq complement component were isolated by collagenase digestion of purified Clq followed by gel filtration. They had an apparent mol. wt of 37,000 as determined by gel filtration. The globular sub-units were found to react with IgG immune complexes as shown by their ability to inhibit the reaction of the latter with 125I-Clq; the functional affinity constant for the sub-unit-IgG complex reaction was estimated to be 1.8–5.8 × 10 4 M −1, values similar to that previously found for the reaction between monomeric IgG and intact Clq. Clq thus binds to immune complexes through the peripheral globular sub-units.

The Mechanism of Activation of the First Component of Complement by a Univalent Hapten-IgG Antibody Complex

Abstract We have previously shown that the univalent hapten, 2,4-dinitrophenyl (DNP)-polylysine when bound to anti-DNP-IgG antibody, yields a 7S complex which is a powerful activator of the classical pathway (Goers et al., 1975, J. Biol. Chem., 250:4918). In this paper we compare this univalent hapten-antibody complex with unassociated antibody, reduced and alkylated antibody, and IgG to which polylysine has been covalently attached. Of these, only the univalent hapten-antibody complex fixes complement. However, both antibody and reduced and alkylated antibody bind equally well to the C1q subunit of the first component, whereas only the univalent hapten-antibody complex shows enhanced binding to the intact first component, C1. Moreover, the antibody-hapten complex or hapten alone will bind to activate C1r,C1s to give C1̄r,C1̄s. The C1s, by itself, is not activated by hapten. Moreover, reassociated C1, composed of C1q,C1r,C1s, is activated by the hapten-antibody complex but not by hapten alone. These results allow us to postulate the existence of a polylysine-sensitive activation site on C1r,C1s which is concealed when C1r, C1s is associated with C1q. Furthermore, we suggest that the binding of hapten-antibody complex to C1 induces a conformational change which causes this site to become exposed. Finally, since all of the substances tested bind equally well to C1q, but only the hapten-antibody complex activates C1, we suggest that peripheral subunits on C1q serve only as anchors to bind C1 to antibody-coated cell surfaces or immune complexes and are not directly involved in the C1 activation process. Activation may occur, we speculate, when the antibody-hapten complex combines with the central stalk of C1q,C1r,C1s.

Isolation of the Subunits of Transcarboxylase and Reconstitution of the Active Enzyme from the Subunits

Abstract The separation of the 12 SH central subunit, the 5 SE peripheral metallo subunit, and the 1.3 SE biotinyl carboxyl carrier protein which are formed on the dissociation of transcarboxylase has been accomplished by molecular sieving on Bio-Gel. The 12 SH and 5 SE subunits have been obtained in nearly homogeneous form as judged by the sedimentation velocity profiles and by acrylamide gel electrophoresis. The 1.3 SE carboxyl carrier protein has given less consistent results; sometimes a single band of molecular weight of approximately 12,000 is obtained on gel electrophoresis in sodium dodecyl sulfate but sometimes there is an additional band of lower molecular weight of approximately 11,000. This lower molecular weight component may result from limited proteolytic degradation in spite of efforts to prevent it. Two or more bands are obtained in the absence of dodecyl sulfate. This heterogeneity may result from aggregation. subunits. The most effective reconstitution is accomplished by a two-step process. First, the 1.3 SE carboxyl carrier protein and 5 SE metallo subunit are combined to form the 6 SE complex; then this product is combined with the 12 SH subunit to yield active enzyme. With a limiting amount of the 6 SE complex and an excess of the 12 SH subunit the resulting enzymatic activity is proportional to the concentration of 6 SE complex. Likewise, with an excess of the 6 SF, complex and the 12 SH subunit limiting, the enzymatic activity is proportional to the concentration of the 12 SH subunit. The maximum specific activities of the reconstituted 6 SE complex and the 12 SH subunit were approximately 60% and 50%, respectively of the value estimated for their specific activities in the native 18 S form of the enzyme with three peripheral 6 SE subunits. Because assay of the 6 SE complex is done using an excess of the 12 SH subunit, this may yield enzyme with a single peripheral subunit. The 6 SE subunit may be less effective in this form. In the case of the 12 SH subunit, the activity is most likely low because the reconstituted 6 SE complex does not contain the full complement of 1.3 SE biotinyl carboxyl carrier proteins. The carboxyl carrier protein provides the groups which link together the 5 SE peripheral subunits with the central subunit. Evidence is presented that the 1.3 SE biotinyl carboxyl carrier must first combine with the 5 SE subunit to assume a form which effectively provides the combining groups for association with the 12 SH subunit.

Chemical analysis and electron microscopy studies of human C1q prepared by different methods.

Five differently isolated and purified human C1q preparations were examined by electron microscopy and analyzed by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate and 0.5 M urea. The amino acid and carbohydrate composition of C1q purified by the DNA method are reported and compared with results obtained on C1q isolated by other procedures. Electron microscopy showed that all C1q preparations had six peripheral subunits connected by fibrillar strands to a central subunit. The presence of small amounts of dimers was also observed. The physico-chemical properties of the molecule are independent of the purification method used. The five C1q preparations labeled with 125I in presence of lactoperoxidase formed two types of noncovalently linked subunits. In each case the smaller (central) subunit contained over thirty times as much radioactivity as the larger (peripheral) subunit supposed to interact with immune complexes. Reduction and alkylation confirmed for each preparation the presence of three polypeptide chains, the smaller of which contained essentially all radioactivity.

Enzymatic Digestion of the First Component of Human Complement (Clq)

Abstract Human C1q was shown to be sensitive to digestion by trypsin, chymotrypsin and collagenase, but not by elastase. Treatment with trypsin and chymotrypsin caused a rapid loss of the hemolytic activity of C1q and its ability to fix to immune complexes. Collagenase digestion of C1q resulted in a rapid loss of the hemolytic activity also, whereas the ability to bind to immune complexes decreased slowly and remained at a constant value of 15%. In contrast to trypsin and chymotrypsin, collagenase treatment of 125I-labeled C1q led to well defined fragments, only some of which were labeled. Adsorption of collagenase-digested C1q on ovalbumin-antiovalbumin complexes indicated the larger non-covalently bound peripheral subunits of C1q to be involved in fixation to immune complexes. None of the fragments was able to initiate lysis in the presence of RC1q. Immunologic analysis of the digest revealed at least two different fragments with respect to antigenicity.

Transcarboxylase: XI. ELECTRON MICROSCOPY AND SUBUNIT STRUCTURE

Abstract The molecular weights of transcarboxylase and its constituent subunits have been determined by sedimentation equilibrium and by gel electrophoresis and the organization of the subunits has been deduced from electron micrographs. The intact enzyme (molecular weight ∼ 792,000, s20,w ∼ 18 S) presents a profile with a large central subunit to which are attached on one side three smaller peripheral subunits. The isolated central subunit (s20,w ∼ 12 S, molecular weight ∼ 360,000) presents two profiles in the electron microscope, one circular (100 A) with a central hole, the other rectangular (70 x 100 A). It dissociates further at pH 9 to give three subunits (s20,w ∼ 6 S, molecular weight ∼ 120,000) with roughly square profiles (70 x 70 A). In sodium dodecyl sulfate, it gives peptide chains of molecular weight 60,000; therefore, it is proposed that the central subunit is a trimer (symmetry C3) of the 6 S subunits each containing two peptide chains. The three smaller peripheral structures (s20,w ∼ 6 S, molecular weight ∼ 144,000) show 55 x 90 A profiles. Each consists of two metal-containing (cobalt, zinc) peptides of 60,000 molecular weight as well as two carboxyl carrier proteins (s20,w ∼ 1.3 S, molecular weight ∼ 12,000) containing biotin. Thus, transcarboxylase is made up of 18 peptides, 6 metals, and 6 biotins. The three peripheral subunits appear to be attached to the central subunit by an extended peptide chain of about 10 to 15 residues, most likely provided by the carboxyl carrier protein. The flexibility of the structure and the lability of the linkage account for the large variety of profiles observed in the electron microscope. Under mild conditions the enzyme dissociates to a modified enzyme of s20,w ∼ 16 S by loss of one of the peripheral subunits. This 16 S component resembles the silhouette of the head of Mickey Mouse in the electron microscope. The intact transcarboxylase rarely shows 3-fold symmetry but this symmetry is seen in complexes of avidin with transcarboxylase in which the central subunit is surrounded by 3 molecules of avidin alternating with the 3 peripheral subunits. The location of the avidin molecules at the ends of the long axis of the peripheral subunit suggests that the carboxyl carrier proteins are also in these positions and extend to one surface of the central subunit. Reconstitution of the transcarboxylase from the isolated central subunit and the peripheral subunit gives active enzyme with typical profiles of transcarboxylase. However, when the enzyme is dissociated in the absence of glycerol and is then reconstituted at pH 5, a new enzymatically active product (s20,w ∼ 24 S) is formed which has two sets of peripheral subunits on the opposite faces of the central subunit, suggesting that by this treatment, additional binding sites for the peripheral subunit have been exposed on the central subunit.

An electron microscope study of the dimorphic spermatozoa of Asplanchna (Rotifera). II. The development of "atypical spermatozoa".

1. The rigid rod develops in atypical germ cells during late stages of rotifer embryonic life. 2. The Golgi system is closely related with the developing rod and appears to contribute to it a dense, homogeneous material via a vesicle fusion process. 3. The completed rod appears to be extruded from the mother cell with a coating of cell membrane in addition to the limiting membrane of the rod. 4. The internal structure of the rod is composed of densely packed microtubules of the 200 A variety oriented parallel to the rods long axis. The microtubules are composed of small tubular peripheral subunits. 5. The classical concept of the dimorphism of rotifer sperm (at least in Asplanchna) must be modified as the so-called “rod spermatozoon” is not a spermatozoon nor even a cell, but a microtubule-filled cellular product. The actual atypical cell is the one which produces the rod in late embryonic life and then apparently degenerates upon completion of the rod structure and its extrusion.