Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi=9.9 and migrates in SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of 15015 and 16296 Da for alpha and beta subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A, B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds to platelet GPIb.     

Dissecting protein‐protein interactions in proteasome assembly: Implication to its self‐assembly

The 26S proteasome is a multi‐catalytic ATP‐dependent protease complex that recognizes and cleaves damaged or misfolded proteins to maintain cellular homeostasis. The 26S subunit consists of 20S core and 19S regulatory particles. 20S core particle consists of a stack of heptameric alpha and beta subunits. To elucidate the structure‐function relationship, we have dissected protein‐protein interfaces of 20S core particle and analyzed structural and physiochemical properties of intra‐alpha, intra‐beta, inter‐beta, and alpha‐beta interfaces. Furthermore, we have studied the evolutionary conservation of 20S core particle. We find the size of intra‐alpha interfaces is significantly larger and is more hydrophobic compared with other interfaces. Inter‐beta interfaces are well packed, more polar, and have higher salt‐bridge density than other interfaces. In proteasome assembly, residues in beta subunits are better conserved than alpha subunits, while multi‐interface residues are the most conserved. Among all the residues at the interfaces of both alpha and beta subunits, Gly is highly conserved. The largest size of intra‐alpha interfaces complies with the hypothesis that large interfaces form first during the 20S assembly. The tight packing of inter‐beta interfaces makes the core particle impenetrable from outer wall of the cylinder. Comparing the three domains, eukaryotes have large and well‐packed interfaces followed by archaea and bacteria. Our findings provide a structural basis of assembly of 20S core particle in all the three domains of life.     

Immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) in the regenerating lizard epidermis indicates a new process for the differentiation of the epidermis in lepidosaurians

The process of keratinocyte differentiation was analyzed in the regenerating epidermis of the lizard Anolis carolinensis, where the genes coding for beta‐proteins (beta‐keratins) are known. The regenerating epidermis forms all epidermal layers found in normal scales (Oberhäutchen‐, beta‐, mesos‐, and alpha‐layer). Three specific proteins representing the larger families of beta‐proteins, glycine‐rich (HgG5, 28% glycine, 3.6% cysteine), glycine‐cysteine medium‐rich (HgGC10, 13% glycine, 14.5% cysteine), and glycine‐cysteine rich (HgGC3, 30.4% glycine, 8.7% cysteine) have been immunolocalized at the ultrastructural level. HgG5 is only present in differentiating beta‐cells, a weak or no labeling is observed in Oberhäutchen and is absent in alpha‐cells. The protein is located in the pale corneous material forming the compact beta‐layer but is absent in mature Oberhäutchen cells. HgGC10 is present among beta‐packets in Oberhäutchen and beta‐cells but disappears in more compact and electron‐pale corneous material. The labeling disappears in mesos‐cells and is present with variable intensity in alpha‐cells, whereas lacunar and clear‐cells are low labeled to unlabeled. HgGC3 is sparse or absent in beta‐cells but is lightly present in the darker corneous material of differentiating and mature alpha‐cells, lacunar‐cells, and clear‐cells. The study suggests that while glycine‐rich proteins (electron‐pale) are specifically used for building the resistant and hydrophobic beta‐layer, cysteine–glycine rich proteins (electron‐denser) are used to form the pliable corneous material present in the Oberhäutchen and alpha‐cells. The differential accumulation of beta‐proteins on the alpha‐keratin cytoskeleton scaffold and not the alternance of beta‐ with alpha‐keratins allow the differentiation of different epidermal layers. © 2012 Wiley Periodicals, Inc.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Lebectin and lebecetin, two C-type lectins from snake venom, inhibit alpha5beta1 and alphaV-containing integrins.

Integrins are essential protagonists in the complex multistep process of cancer progression and metastasis. We recently reported that lebectin, a novel C-type lectin from Macrovipera lebetina venom, displays an anti-integrin activity. In this study, we extend this observation to lebecetin, a second C-type lectin isolated from the same venom and previously reported as a potent inhibitor of platelet aggregation. Both venom lectins appear to exert their effect on cell adhesion, migration, invasion and proliferation by inhibiting alpha5beta1 and alphav-containing integrins. Moreover, the inhibition of alpha5beta1 and alphav integrins is likely due to the binding of venom peptides, as both lebectin and lebecetin co-immunoprecipitate with these integrins. Lebectin and lebecetin are thus the first examples of venom C-type lectins inhibiting an integrin other than the collagen receptor alpha2beta1.     

Immunolocalization of keratin‐associated beta‐proteins in developing epidermis of lizard suggests that adhesive setae contain glycine–cysteine‐rich proteins

The localization of specific keratin‐associated beta‐proteins (formerly referred to as beta‐keratins) in the embryonic epidermis of lizards is not known. Two specific keratin‐associated beta‐proteins of the epidermis, one representing the glycine‐rich subfamily (HgG5) and the other the glycine‐cysteine medium‐rich subfamily (HgGC10), have been immunolocalized at the ultrastructural level in the lizard Anolis lineatopus. The periderm and granulated subperiderm are most immunonegative for these proteins. HgG5 is low to absent in theOberhäutchen layer while is present in the forming beta‐layer, and disappears in mesos‐ and alpha‐layers. Instead, HgGC10 is present in the Oberhäutchen, beta‐, and also in the following alpha‐layers, and specifically accumulates in the developing adhesive setae but not in the surrounding cells of the clear layer. Therefore, setae and their terminal spatulae that adhere to surfaces allowing these lizards to walk vertically contain cysteine–glycine rich proteins. The study suggests that, like in adult and regenerating epidermis, the HgGC10 protein is not only accumulated in cells of the beta‐layer but also in those forming the alpha‐layer. This small protein therefore is implicated in resistance, flexibility, and stretching of the epidermal layers. It is also hypothesized that the charges of these proteins may influence adhesion of the setae of pad lamellae. Conversely, glycine‐rich beta‐proteins like HgG5 give rise to the dense, hydrophobic, and chromophobic corneous material of the resistant beta‐layer. This result suggests that the differential accumulation of keratin‐associated beta‐proteins over the alpha‐keratin network determines differences in properties of the stratified layers of the epidermis of lizards. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Membrane attachment of recombinant G-protein alpha-subunits in excess of beta gamma subunits in a eukaryotic expression system

Recombinant cDNAs encoding the alpha-subunits of Gi1, Gi2, Gi3, Go and Gs were transfected into COS cells with the pCD-PS mammalian expression vector. Expression of each G alpha was verified using subtype-specific peptide antisera on immunoblots. Quantitative immunoblotting of alpha and beta subunits indicated: i) that there was no change in expression of endogenous beta subunits, and ii) overexpression of alpha subunits could achieve a ratio of alpha:beta greater than 25:1. Despite the excess of alpha over beta, the G alpha subunits were found predominantly in the membrane fraction. The results demonstrate that G alpha subunits can attach to the membrane independently of beta gamma subunits.     

Immunolocalization of large corneous beta‐proteins in the green anole lizard (Anolis carolinensis) suggests that they form filaments that associate to the smaller beta‐proteins in the beta‐layer of the epidermis

The distribution of large corneous beta‐proteins of 18–43 kDa (Ac37, 39, and 40) in the epidermis of the lizard Anolis carolinensis is unknown. This study analyses the localization of these beta‐proteins in different body scales during regeneration. Western blot analysis indicates most protein bands at 40–50 kDa suggesting they mix with alpha‐keratin of intermediate filament keratin proteins. Ac37 is present in mature alpha‐layers of most scales and in beta‐cells of the outer scale surface in some scales but is absent in the Oberhäutchen, in the setae and beta‐layer of adhesive pads and in mesos cells. In differentiating beta‐keratinocytes Ac37 is present over 3–4 nm thick filaments located around the amorphous beta‐packets and in alpha‐cells, but is scarce in precorneous and corneous layers of the claw. Ac37 forms long filaments and, therefore, resembles alpha‐keratins to which it probably associates. Ac39 is seen in the beta‐layer of tail and digital scales, in beta‐cells of regenerating scales but not in the Oberhäutchen (and adhesive setae) or in beta‐ and alpha‐layers of the other scales. Ac40 is present in the mature beta‐layer of most scales and dewlap, in differentiating beta‐cells of regenerating scales, but is absent in all the other epidermal layers. The large beta‐proteins are accumulated among forming beta‐packets of beta‐cells and are packed in the beta‐corneous material of mature beta‐layer. Together alpha‐keratins, large beta‐proteins form the denser areas of mature beta‐layer that may have a different consistence that the electron‐paler areas. J. Morphol. 276:1244–1257, 2015. © 2015 Wiley Periodicals, Inc.     

Individual embryonic fibroblasts express multiple beta chains in association with the alpha v integrin subunit. Loss of beta 3 expression with cell confluence.

The alpha chain of the vitronectin receptor, alpha v, has been found in association with the integrin subunits beta 1, beta 3, or beta 5 on different cell types. We show here that cultured embryonic fibroblasts simultaneously display alpha v beta 3, alpha v beta 1, and alpha v in association with two other beta subunits, one of which is probably beta 5. Polymerase chain reaction analysis of single cells isolated by micromanipulation identified mRNA for alpha v, beta 1, beta 3, and beta 5 in six of eight clones. Immunoprecipitation of iodinated cell surface proteins with a monoclonal antibody to alpha v indicated that the relative proportions of the different beta chains in association with alpha v varied, particularly between two different cell lines. The cytokines platelet-derived growth factor, transforming growth factor beta 1, and tumor necrosis factor alpha did not appear to alter this ratio although tumor necrosis factor alpha increased the surface expression of the alpha v-associated integrins; but overnight culture in basic fibroblast growth factor caused a lower expression of alpha v beta 1 and alpha v beta 5 with no reduction in alpha v beta 3 expression. When the cell cultures were grown to complete confluence, surface expression of beta 3 was abolished, and the expression of an unknown beta chain (beta u) became more prominent. This effect was not overcome by culturing confluent cells with basic fibroblast growth factor. Affinity column chromatography showed that alpha v beta 5 bound to vitronectin but alpha v beta 1 did not, whereas alpha v beta 1 but not alpha v beta 5 bound to fibronectin. These results suggest that, on individual cells, the beta subunits found in association with alpha v may vary according to the proliferative capacity of the cell and that the promiscuous beta 3 subunit is progressively replaced by beta subunits of individual ligand specificity.     

Learning‐dependent regulation of beta2 and gamma2 subunits of GABAAR mRNA in sensory cortex of mice

GABAA receptors are heteromeric, ligand‐gated ion channels, built up with 19 different subunits. In the somatosensory cortex the most prevalent subunits are: alpha1, beta2 and gamma2. We showed that classical conditioning, in which stimulation of a row of vibrissae (CS) was paired with a tail shock (UCS), results in changes in cortical inhibitory system. Among others, GABAAalpha1 mRNA is up‐regulated in layer IV of cortical representation of trained row of vibrissae. We examined the mRNA expression level of beta2 and gamma2 subunits of GABAAR. For in situ hybridization, 35S‐labeled oligonucleotides were used as antisense probes. The effects were examined 1 h, 24 h and 5 days after the training lasting 3 days. There are no changes observed in beta2 subunits mRNA level. Expression of mRNA of gamma2 subunits increased 5 days after the training, but in contrast to other elements of gabaergic system we investigated, the change was observed in layers II/III. This indicates that learning‐dependent regulation of GABAA receptor phenotype is specific for given neuronal subtype.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

chlC (nar) operon of Escherichia coli includes structural genes for alpha and beta subunits of nitrate reductase.

The synthesis of the alpha and beta subunits of nitrate reductase by 20 chlC::Tn5 insertion mutants of Escherichia coli was determined by immune precipitation of the subunits from fractions of cell extracts. Only two of the mutants produced either subunit in detectable amounts; these two accumulated the alpha subunit, but no beta subunit. In both cases the alpha subunit was present in the cytosolic fraction, in contrast to wild-type cells, in which both subunits are present mainly in the membrane fraction. EcoRI restriction fragments containing the Tn5 inserts from five of the mutants were cloned into pBR322. The insertions were localized on two contiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped the contiguous ends of the two fragments. An insertion that permitted alpha subunit formation defined one end of the 5.6-kilobase region. The results indicated that the genes encoding the alpha and beta subunits of nitrate reductase were part of a chlC (nar) operon that is transcribed in the direction alpha leads to beta.     

Functional properties of recombinant rat GABAA receptors depend upon subunit composition.

GABA-gated chloride channels were expressed in human embryonic kidney cells following transfection of cDNAs encoding the alpha 1, beta 2, and gamma 2 subunits of the rat GABAA receptor (GABAR). Functional properties were determined using patch-clamp techniques in the whole-cell and outside-out configurations. Large whole-cell currents were observed in cells expressing the alpha 1 beta 2, alpha 1 gamma 2, and alpha 1 beta 2 gamma 2 subunit combinations. The unique characteristics of GABAR channels consisting of these subunit combinations depended upon the presence or absence of beta 2 and gamma 3 subunits. GABA-activated currents in cells expressing GABARs with the beta 2 subunit desensitized faster and showed greater outward rectification, and the channels had a shorter mean open time than GABARs composed of alpha 1 gamma 2 subunits. When the gamma 2 subunit was present the resulting GABAR channels had a larger conductance. The slope of the concentration-response curve was significantly steeper for GABARs composed of alpha 1 beta 2 gamma 2 subunits compared with GABARs consisting of alpha 1 beta 2 or alpha 1 gamma 2 subunit combinations.     

The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron

We studied the distribution of the alpha 1-alpha 6 subunits of beta 1 integrins in developing and adult human kidney using a panel of mAbs in indirect immunofluorescence microscopy. Uninduced mesenchyme displayed a diffuse immunoreactivity for only the alpha 1 integrin subunit. At the S-shaped body stage of nephron development, several of the alpha subunits were characteristically expressed in distinct fetal nephron segments, and the pattern was retained also in the adult nephron. Thus, the alpha 1 subunit was characteristically expressed in mesangial and endothelial cells, the alpha 2 in glomerular endothelium and distal tubules, the alpha 3 in podocytes, Bowman's capsule, and distal tubules, and the alpha 6 subunit basally in all tubules, and only transiently in podocytes during development. Unlike the alpha 3 and alpha 6 subunits, the alpha 2 subunit displayed an overall cell surface distribution in distal tubules. It was also distinctly expressed in glomerular endothelia during glomerulogenesis. The beta 4 subunit was expressed only in fetal collecting ducts, and hence the alpha 6 subunit seems to be complexed with the beta 1 rather than beta 4 subunit in human kidney. Of the two fibronectin receptor alpha subunits, alpha 4 and alpha 5, only the latter was expressed, confined to endothelia of developing and adult blood vessels, suggesting that these receptor complexes play a minor role during nephrogenesis. The present results suggest that distinct integrins play a role during differentiation of specific nephron segments. They also indicate that alpha 3 beta 1 and alpha 6 beta 1 integrin complexes may function as basement membrane receptors in podocytes and tubular epithelial cells.     

Formation of adherens and communicating junctions coordinate the differentiation of the shedding‐layer and beta‐epidermal generation in regenerating lizard epidermis

In the lizard epidermis, the formation of a stratified alpha‐ and beta‐layer, separated by a shedding complex for molting, suggests that keratinocytes communicate in a coordinated manner after they leave the basal layers during the shedding cycle. I have therefore studied the localization of cell junctional proteins such as beta‐catenin and connexins 43 and 26 during scale regeneration in lizard using immunocytochemistry. Beta‐catenin is also detected in nuclei of basal cells destined to give rise to the Oberhäutchen and beta‐cells suggesting activation of the Wnt‐pathway during beta‐cell differentiation. The observations show that cells of the entire shedding layer (clear and Oberhäutchen) and beta‐layer are connected by beta‐catenin (adherens junctions) and connexins (communicating junctions) during their differentiation. This likely cell coupling determines the formation of a distinct shedding and beta‐layer within the regenerating epidermis. The observed pattern of cell junctional stratification suggests that after departing from the basal layer Oberhäutchen and beta‐cells form a continuous communicating compartment that coordinates the contemporaneous differentiation along the entire scale. While the beta‐layer matures the junctions are lost while other cell junctions are formed in the following mesos‐ and alpha‐cell layers. This process determines the formation of layers with different texture (harder or softer) and the precise localization of the shedding layer within lizard epidermis. J. Morphol. 275:693–702, 2014. © 2014 Wiley Periodicals, Inc.     

Unequal synthesis and differential degradation of propionyl CoA carboxylase subunits in cells from normal and propionic acidemia patients.

We have characterized further the molecular basis of human inherited propionyl CoA carboxylase deficiency by measuring steady state levels of the mRNAs coding for the enzyme's two protein subunits (alpha and beta) and by estimating initial synthesis and steady state levels of the protein subunits in skin fibroblasts from controls and affected patients. We studied cell lines from both major complementation groups (pccA and pccBC) corresponding, respectively, to defects in the carboxylase's alpha and beta subunits. Analysis of pccA lines revealed the absence of alpha chain mRNA in three and an abnormally small alpha-mRNA in a fourth. Despite the presence of normal beta-mRNA in each of these pccA lines, there was complete absence of both alpha and beta protein subunits under steady state conditions, even though new synthesis and mitochondrial import of beta precursors was normal. Results in nine pccBC lines revealed normal alpha mRNA in each, while the amounts of beta-mRNA were distinctly reduced in every case. Correspondingly, alpha protein subunits were present in normal amounts at steady-state, but beta subunits were uniformly decreased. In addition, in six of the nine beta deficient cell lines, partially degraded beta-subunits were observed. To help interpret these results, synthesis and stability of carboxylase subunits were studied in intact HeLa cells using a pulse-chase protocol. Whereas alpha chains were stable over the four hour interval studied, beta chains--initially synthesized in large excess over alpha chains--were degraded rapidly reaching equivalence with alpha chains after two hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.