Conversion of the alpha component of penicillin-binding protein 1b to the beta component in Escherichia coli.

Among components alpha, beta, and gamma of penicillin-binding protein 1b, the alpha and gamma components were confirmed to represent the primary gene products by agreement of their N-terminal amino acid sequences with those predicted from the nucleotide sequence of the ponB (penicillin-binding protein 1b) gene with exclusion of the first methionine in each component. The generation of beta occurred primarily after cell disruption, and the simultaneous loss of alpha suggested the conversion of alpha to beta. The N-terminal amino acid sequence analyzed for beta showed that the conversion was due to the removal of 24 amino acids from the N terminus of alpha.     

Chloroplast translational initiation factor 3. Purification and characterization of multiple forms from Euglena gracilis.

The chloroplast translational initiation factor 3 (IF-3chl) has been purified by a combination of gravity and high pressure liquid chromatographic steps. IF-3chl activity has been resolved into three forms designated alpha, beta, and gamma. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the alpha form corresponds to a single polypeptide with a molecular mass of approximately 34 kDa. The beta and gamma forms have been purified to near homogeneity, and both forms appear to function as monomers with molecular masses of about 39-42 kDa. All three forms are heat stable. All the forms of IF-3chl detected enhance the poly (A,U,G)-dependent binding of the initiator tRNA to chloroplast 30 S ribosomal subunits in the presence of Escherichia coli IF-1 and IF-2. The chloroplast factor, unlike the corresponding bacterial factor, does not have a strong RNA binding activity.     

Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase

Ten, electrophoretically distinct, molecular forms of alcohol dehydrogenase have been isolated from a single human liver by affinity and ion-exchange chromatography. The starch gel electrophoresis patterns after the dissociation-recombination of the forms are consistent with the hypothesis that they arise from the random combination of alpha, beta 1, gamma 1, and gamma 2 subunits into six heterodimeric and four homodimeric isoenzymes. Large differences in kinetic properties are observed for the homodimeric isoenzymes, alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. At pH 7.5, the Km value of beta 1 beta 1 for ethanol is 0.049 mM and that of alpha alpha is 4.2 mM. Forms gamma 1 gamma 1 and gamma 2 gamma 2 do not obey Michaelis-Menten kinetics at pH 7.5 but exhibit negative cooperativity with Hill coefficients of 0.54 and 0.55 and [S]0.5 values of 1.0 and 0.63 mM, respectively. However, all isoenzymes display Michaelis-Menten kinetics for ethanol oxidation at pH 10.0 with Km values ranging from 1.5 to 3.2 mM. The maximum specific activity of beta 1 beta 1 is considerably lower than that of the other three homodimers at both pH 7.5 and 10.0. The Km values of the four homodimers for NAD+ at pH 7.5 range from 7.4 to 13 microM and those for NADH, from 6.4 to 33 microM. Ki values for NADH range from 0.19 to 1.6 microM. At pH 7.5, the kinetic properties of alpha alpha and beta 1 beta 1, prepared in vitro from dissociated and recombined alpha beta 1, are similar to those of the native homodimers. The forms gamma 1 gamma 1 and gamma 2 gamma 2, prepared from dissociated and recombined alpha gamma 1 and beta 1 gamma 2, respectively, exhibit negative cooperativity with Hill coefficients that are similar to those seen with the respective native homodimers.     

The human LT serum. X. The initial form released by T-enriched lymphocytes is 150,000 m.w., associated with small nonlytic components, and can dissociate into the smaller alpha, beta, and gamma m.w. classes

The present studies examine the various lymphotoxin (LT) forms released in vitro by phytohemagglutinin- (PHA) activated T-enriched (Te) human peripheral blood lymphocytes. It is clear that Te cells rapidly released (24 to 48 hr) these molecules in vitro. The 1st cell-lytic form detected in these supernatants is a 140-160,000 m.w. molecule(s) termed precursor alpha heavy (P alpha H). This form does not express alpha-LT antigenic determinants but is neutralized by antisera from animals injected with serum-free PHA-activated unseparated lymphocyte supernatants (anti-WS). The P alpha H is converted into alpha H, which expresses alpha determinants, by passage through molecular sieving columns or by treatment with low levels of Nonidet P-40 or urea. These treatments dissociate a small nontoxic 10-20,000 m.w. molecule(s), termed precursor factor (Pf), which masks the alpha-LT determinant on the P alpha H molecule. The dissociation of Pf is reversible, since alpha H from the molecular sieving columns will reassociate with the Pf. The alpha H LT class can further dissociate into the smaller alpha, beta, and gamma LT forms upon chromatography on a molecular sieving column, and a certain small percentage of the alpha H forms appear capable of associating to form the high m.w. complex (Cx) LT class. These findings suggest P alpha H may represent an intermediate that requires additional processing in order to proceed down 1 of 2 pathways: a) formation of complexes that are highly cell-lytic, or b) degradation by dissociation into the smaller weakly cell-lytic molecules identified as LT forms.     

Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana

The proprotein precursors of storage proteins are post-translationally processed to produce their respective mature forms within the protein storage vacuoles of maturing seeds. To investigate the processing mechanism in vivo, we isolated Arabidopsis mutants that accumulate detectable amounts of the precursors of the storage proteins, 12 S globulins and 2 S albumins, in their seeds. All six mutants isolated have a defect in the beta VPE gene. VPE (vacuolar processing enzyme) is a cysteine proteinase with substrate specificity toward an asparagine residue. We further generated various mutants lacking different VPE isoforms: alpha VPE, beta VPE, and/or gamma VPE. More than 90% of VPE activity is abolished in the beta vpe-3 seeds, and no VPE activity is detected in the alpha vpe-1/beta vpe-3/gamma vpe-1 seeds. The triple mutant seeds accumulate no properly processed mature storage proteins. Instead, large amounts of storage protein precursors are found in the seeds of this mutant. In contrast to beta vpe-3 seeds, which accumulate both precursors and mature storage proteins, the other single (alpha vpe-1 and gamma vpe-1) and double (alpha vpe-1/gamma vpe-1) mutants accumulate no precursors in their seeds at all. Therefore, the vegetative VPEs, alpha VPE and gamma VPE, are not necessary for precursor processing in the presence of beta VPE, but partly compensates for the deficiency in beta VPE in beta vpe-3 seeds. In the absence of functional VPEs, a proportion of pro2S albumin molecules are alternatively cleaved by aspartic proteinase. This cleavage by aspartic proteinase is promoted by the initial processing of pro2S albumins by VPE. Our overall results suggest that seed-type beta VPE is most essential for the processing of storage proteins, and that the vegetative-type VPEs and aspartic proteinase complement beta VPE activity in this processing.     

Biosynthesis and maturation of the Lyt-2/3 molecular complex in mouse thymocytes

The biosynthesis and maturation of the three subunits alpha (Mr = 37,000), beta (Mr = 32,000), and gamma (Mr = 27,000) of the mouse Lyt-2/3 antigenic complex have been studied by using two monoclonal antibodies directed against a monomorphic determinant of the Lyt-2 antigen. Short time-pulse labeling of thymocytes reveals three different high mannose intermediates that give rise upon endo-beta-N-acetyl-glucosaminidase H digestion to three distinct precursor polypeptides of Mr = 22,000 (alpha P), Mr = 18,000 (beta P), and Mr = 19,500 (gamma P). Pulse-chase analysis indicates rapid posttranslational processing, because mature forms already appear after 10 min of chase. The half-life of the endo-H-sensitive early forms are in the range of 20 to 30 min. Both the alpha and beta subunits are suggested to contain three N-asparagine-linked oligosaccharides, one of which is of the high mannose type. In contrast, the gamma-chain contains only one such glycan unit of the complex type. Moreover, the results presented show that all three chains undergo additional posttranslational modifications. Finally, the data suggest that the cytoplasmic domains of these chains are of different size.     

G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high-affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP-ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases.     

Evidence for the existence of protomers in the assembly of encephalomyocarditis virus.

Two capsid precursor subunits, which sediment on glycerol gradients at 13S and 14S, respectively, have been identified in cytoplasmic extracts of encephalomyocarditis virus-infected HeLa cells. The 13S subunit, which was detected after a 10-min pulse label with -3H-labeled amino acids, contained only capsid precursor chain A (mol wt 100,000). When the 10-min pulse label in such cells was chased for 20 min, the A-containing 13S subunit in the cytoplasmic extracts was replaced by a 14S subunit containing equimolar proportions of three chains: epsilon, gamma, and alpha. This (epsilon, gamma, alpha)-containing 14S subunit could be dissociated into 6S subunits with the same polypeptide composition. The sedimentation properties and the polypeptide stoichiometry of these three precursor subunits, when compared with those of the 13S, (beta, gamma, alpha)(5), and 5S, (beta, gamma, alpha), subunits derived by acid dissociation of purified virions, suggest the following structural assignments: 13S, (A)(5); 14S, (epsilon, gamma, alpha)(5), 6S, (epsilon, gamma, alpha). The molecular weights of the individually isolated capsid chains were determined by gel filtration in 6 M guanidine hydrochloride to be: epsilon, 36,000; alpha, 32,000; beta, 29,500; gamma, 26,500; and delta, 7,800. With the exception of the delta-chain, these values are in reasonable agreement with the values previously determined by electrophoresis on sodium dodecyl sulfatepolyacrylamide gels. These data support the hypothesis that picornavirus capsids are assembled from identical protomers according to the following scheme: (A) leads to (A)(5) leads to (epsilon, gamma, alpha)(5) leads to (delta, beta, gamma, alpha)60-n(epsilon, gamma, alpha)n where n is the number of immature protomers per virion.     

The specificity of biliverdin reductase. The reduction of biliverdin XIII isomers

The substrate specificity of the different molecular forms of biliverdin reductase (bilirubin:NAD(P)+ oxidoreductase, EC 1.3.1.24) using biliverdin XIII alpha, XIII beta and XIII gamma was examined. It was found that molecular form 1 (the major form in normal rat liver) reduced biliverdin XIII alpha at a much higher rate than the other two isomers. Molecular form 2 (the minor form) reduced isomers XIII alpha and XIII beta at similar rates, while molecular form 3 (the major form induced by CoCl2 treatment) reduced the XIII beta isomer at a slightly higher rate than the XIII alpha isomer. Molecular forms 2 and 3, both reduced isomer XIII gamma more slowly than they reduced the XIII alpha and XIII beta isomers. These results are similar to those obtained previously using biliverdins IX alpha, IX beta and IX gamma, suggesting that biliverdin reductase specificity is related to the type of the isomer rather than to the series (IX or XIII) of the isomer.     

Ligand-induced conformational changes in the Escherichia coli F1 adenosine triphosphatase probed by trypsin digestion

Digestion of the F1-ATPase of Escherichia coli with trypsin stimulated ATP hydrolytic activity and removed the delta and epsilon subunits of the enzyme. A species represented by the formula alpha 1(3) beta 1(3) gamma 1, where alpha 1, beta 1 and gamma 1 are forms of the native alpha, beta and gamma subunits which have been attacked by trypsin, was formed by trypsin digestion in the presence of ATP. In the presence of ATP and MgCl2, conversion of gamma to gamma 1 was retarded and the enzyme retained the epsilon subunit. These results imply that binding of ATP to the beta subunits alters the conformation of ECF1 to increase the accessibility of the gamma subunit to trypsin. The likely trypsin cleavage sites in the alpha, beta and gamma subunits are discussed. ECF1 from the alpha subunit-defective mutant uncA401, or after treatment with N,N'-dicyclohexylcarbodiimide or 4-chloro-7-nitrobenzofurazan, was present in a conformation in which the gamma subunit was readily accessible to trypsin and could not be protected by the presence of ATP and MgCl2. In a similar manner to native E. coli F1-ATPase, the hydrolytic activity of the trypsin-digested enzyme was stimulated by the detergent lauryldimethylamine N-oxide. Since the digested enzyme lacked the epsilon subunit, a putative inhibitor of hydrolytic activity, a mechanism for the stimulation which involves loss or movement of this subunit is untenable.     

Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.     

Molecular multiplicity of nuclease TT1 from Thermus thermophilus HB8

Purified nuclease TT1 from Thermus thermophilus HB8 has multimolecular weight forms, each of which is composed of three different subunits, alpha (10.8 x 10(4)), beta (7.8 x 10(4)), and gamma (4.1 x 10(4)). The molecular weights of this enzyme were estimated by gel filtration, polyacrylamide gel electrophoresis and equilibrium sedimentation. It was found that most of the enzyme has a molecular weight of about 22 x 10(4) being a monomer having the subunit composition of alpha beta gamma. The remaining part of the enzyme has larger molecular weights and is considered to be size-isomers of alpha beta gamma. The alpha-helical content, 5.5--6.5%, and the beta-structure, about 28%, were estimated from the CD spectrum at 4 degrees C.     

Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase

Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli. Following transformation of either of these plasmids into E. coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera. Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261, 9587-9590). Expression of these cDNAs in E. coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively. Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E. coli expressing either form of Gs alpha. Partial purification of Gs alpha from E. coli lysates yields preparations in which significant and stable activity can be assayed. Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent. As expressed in E. coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with [32P]NAD+ and cholera toxin. However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E. coli appears to be active. Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity. Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed [32P]ADP-ribosylation.     

Effect of in vitro interferon treatment on the rapid clearance of murine leukemia cells in vivo.

Previous work showed that interferon (IFN) can protect target cells from NK mediated lysis in vitro. In the present study we investigate the effect of IFN alpha/beta or IFN gamma treatment of three different murine leukemia cell lines. For this purpose FLC-745 (susceptible to the antiproliferative activity of IFN alpha/beta and gamma), FLC-3C18 (IFN alpha/beta -resistant and IFN gamma - susceptible) of DBA/2 origin and EL-4 (IFN alpha/beta - susceptible and IFN gamma - resistant) leukemia of C57B1/6 origin were treated with IFN alpha/beta or gamma in vitro and assayed for their susceptibility to natural resistance measured in vivo as organ rapid clearance 4 hr after iv injection into syngeneic mice. Using young or Poly I:C stimulated hosts, but not mice with low levels of natural resistance (i.e. older animals or mice treated with cyclophosphamide), slower elimination of treated cells was observed with: (a) FLC-745 cells treated with IFN alpha/beta and IFN gamma and (b) FLC 3C18 treated with IFN gamma. Such a delayed clearance was not observed with: (a) FLC-3C18 cells treated with IFN alpha/beta and (b) EL-4 leukemia cells preincubated with IFN alpha/beta or IFN gamma. These results suggest that under selected conditions IFNs can protect leukemic cells from in vivo natural reactivity.     

Hybrid ATPase's formed from subunits of coupling factor F1's of Escherichia coli and thermophilic bacterium PS3

ATPase complexes were reconstituted from homologous and heterologous combinations of alpha, beta, and gamma subunits of coupling factor ATPase TF1 of thermophilic bacterium PS3 and EF1 of Escherichia coli. TF1 and alpha beta gamma complex reconstituted from TF1 subunits were thermostable and activated by methanol, sodium dodecyl sulfate and anions and they were halophilic, whereas EF1 and its three-subunit complex did not show these properties. The hybrid ATPase alpha T beta T gamma E (complex of the alpha and beta subunits of TF1 and the gamma subunit of EF1) showed closely similar properties to TF1 except for thermostability, while alpha E beta E gamma T (alpha and beta from EF1 and gamma from TF1) had similar properties to EF1. These results suggest that alpha and/or beta is required for the properties of F1. The complex alpha E beta T gamma E showed similar properties to EF1 except for its optimum pH: this complex had a broad pH optimum at about pH 7, whereas EF1 had an optimum at pH 8.5. No hybrid complexes were thermostable, suggesting that all three subunits of TF1 are required for heat stability. These hybrids showed higher halophilicity than EF1, although they were less halophilic than TF1. The hybrid enzymes studied here are the first thermophilic-mesophilic hybrid enzymes obtained.     

An indel within the C8 alpha subunit of human complement C8 mediates intracellular binding of C8 gamma and formation of C8 alpha-gamma

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex, or MAC. It is an oligomeric protein composed of three subunits (C8alpha, C8beta, C8gamma) that are products of different genes. In C8 from serum, these are arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. In this study, the site on C8alpha that mediates intracellular binding of C8gamma to form C8alpha-gamma was identified. From a comparative analysis of indels (insertions/deletions) in C8alpha and its structural homologues C8beta, C6, C7, and C9, it was determined that C8alpha contains a unique insertion (residues 159-175), which includes Cys(164) that forms the disulfide bond to C8gamma. Incorporation of this sequence into C8beta and coexpression of the resulting construct (iC8beta) with C8gamma produced iC8beta-gamma, an atypical disulfide-linked dimer. In related experiments, C8gamma was shown to bind noncovalently to mutant forms of C8alpha and iC8beta in which Cys(164)-->Gly(164) substitutions were made. In addition, C8gamma bound specifically to an immobilized synthetic peptide containing the mutant indel sequence. Together, these results indicate (a) intracellular binding of C8gamma to C8alpha is mediated principally by residues contained within the C8alpha indel, (b) binding is not strictly dependent on Cys(164), and (c) C8gamma must contain a complementary binding site for the C8alpha indel.     

Monoclonal antibodies to Escherichia coli F1-ATPase. Correlation of binding site location with interspecies cross-reactivity and effects on enzyme activity

Twenty-one hybridoma cell lines which secret antibodies to the subunits of the Escherichia coli F1-ATPase were produced. Included within the set are four antibodies which are specific for alpha, six for beta, three for gamma, four for delta and four for epsilon. The antibodies were divided into binding competition subgroups. Two such competition subgroups are represented for the alpha, beta, and epsilon subunits, one for delta and three for gamma. The ability to bind intact F1-ATPase was demonstrated for some of the antibodies to alpha and beta, and for all of those to delta, while the antibodies to gamma and epsilon gave unclear results. All of the antibodies to alpha and beta which bound ATPase were found to have effects on the ATPase activity of purified E. coli F1-ATPase. One of those to alpha inhibited activity by about 30%. Another anti-alpha was mildly stimulatory. The four antibodies to beta which bound ATPase inhibited activity by 90%. In contrast, membrane-bound ATPase was hardly affected by the antibodies to alpha, but was inhibited by 40-60% by the antibodies to beta. The other antibodies to alpha and beta bound only free subunits, or partially dissociated ATPase, suggesting that their epitopes are buried between subunits in ATPase. These antibodies had no effects on activity. The ability of the antibodies to recognize ATPase subunits present in crude extracts from mitochondria, chloroplasts, and a variety of bacteria was tested using nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. One anti-beta specifically recognized proteins in the range of 50,000-60,000 daltons in each of the extracts, although the reaction with mitochondrial beta was weak. Some of the other antibodies had limited cross-reaction, but most were specific for the E. coli protein. In some species, those proteins which were recognized by the anti-beta ran with a higher apparent molecular weight than proteins which were recognized by an anti-alpha. All antibodies which exhibited cross-reactivity were found to recognize sites which were not exposed in intact ATPase, implying that the surfaces which lie between subunits are most highly conserved.