Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers. Demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs.

Novel methods for the isolation of gamma-aminobutyric acidA (GABAA) receptor alpha subunit iso-oligomers have been developed. Thus, populations of GABAA receptors containing the GABAA receptor alpha 1 subunit, the alpha 2 subunit, and the alpha 3 subunit have been purified from sodium deoxycholate extracts of bovine cerebral cortex with the retention of specific [3H]flunitrazepam-binding activity by anti-alpha 1 324-341, anti-Cys alpha 2 414-424, or anti-Cys alpha 3 454-467 antibody affinity chromatography, respectively. The relative abundance of the different specificity alpha subunits in these preparations was compared with benzodiazepine affinity chromatography-purified GABAA receptors by immunoblotting. In each case, it was found that although the immunoreactivity with the specific alpha subunit antibody that was used for purification was enriched in immunoaffinity-purified receptors, reactivity with the other alpha subunit specificity antibodies, together with anti-gamma 2 1-14 Cys immunoreactivity was found. Immunoprecipitation of GABAA receptors purified by anti-alpha 1 324-341 antibody affinity chromatography by all three anti-alpha subunit antibodies employed, together with the use of anti-alpha 1 324-341 and anti-Cys alpha 2 414-424 antibody affinity columns in series, further substantiated the partial co-purification of the different polypeptides. These results demonstrate the copurification of the gamma 2 subunit with each population of alpha 1, alpha 2, alpha 3 subunit-enriched GABAA receptors. They also show the existence of minor populations of GABAA receptors that contain alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs within single oligomers.     

Acid-induced dissociation of alpha A- and alpha B-crystallin homopolymers.

Homopolymers were constructed from the alpha A and alpha B polypeptides isolated from the lens protein alpha-crystallin. As the pH is lowered from 7.0 to 3.4, these homopolymers dissociate to smaller species with molecular masses ranging from 80 to 250 kDa for the alpha A and around 140 kDa for the alpha B dissociation products. The pKa for this dissociation was 3.8 +/- 0.2 for alpha A and 4.1 +/- 0.1 for alpha B homopolymers. Further decreases in pH, to 2.5, resulted in the presence of only denatured alpha B polypeptides, whereas the alpha A dissociation products remained intact. Fractionation of the acid dissociation products from the alpha A homopolymer at pH 2.5 yielded stable species with molecular masses of 220 +/- 30, 160 +/- 20, and 90 +/- 10 kDa. The majority of the population at acid pH consisted of the 160 kDa species. Conformational analysis of these species revealed that most of the secondary structure of the original alpha A homopolymer was retained but that the tertiary structure was perturbed. Fluorescence quenching and energy transfer measurements suggested that the molecule had undergone acid expansion, with the greatest perturbation observed in the smallest particles. The results from this work suggest that alpha A homopolymers are heterogeneous populations of aggregates of a "monomeric" molecule with a molecular mass of 160 kDa. This "monomeric" molecule may be formed from the association of two tetrameric units.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Jellyfish mesogloea collagen. Characterization of molecules as alpha 1 alpha 2 alpha 3 heterotrimers

The mesogloea collagen of a primitive animal, the jellyfish Stomolophus nomurai, belonging to the class Scyphozoa in the Coelenterata, was studied with respect to its chain structure. Most of the mesogloea collagen was solubilized by limited digestion with pepsin and isolated by selective precipitation at 0.9 m NaCl in 0.5 M acetic acid. Upon denaturation, the pepsin-solubilized collagen produced three distinct alpha chains, alpha 1, alpha 2, and alpha 3, in comparable amounts which were separable by CM-cellulose chromatography. The nonidentity of these alpha chains was confirmed by amino acid and carbohydrate analyses and peptide mapping. Furthermore, the introduction of intramolecular cross-links into native molecules by formaldehyde yielded a large proportion of gamma 123 chain with chain structure alpha 1 alpha 2 alpha 3, as judged by chromatographic behavior and peptide maps. We concluded that mesogloea collagen is comprised of alpha 1 alpha 2 alpha 3 heterotrimers and is chemically like vertebrate Type V collagen. On the other hand, sea anemone mesogloea collagen from the class Anthozoa was previously reported to comprise (alpha)3 homotrimers (Katzman, R. L., and Kang, A. H. (1972) J. Biol. Chem. 247, 5486-5489). On the basis of these findings, we assume that alpha 1 alpha 2 alpha 3 heterotrimers arose in evolution with the divergence of Scyphozoa and Anthozoa.     

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.     

Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes.

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.     

Polymorphism of human alpha fucosidase.

A common polymorphism of human alpha fucosidase has been detected by the technique of isoelectric focusing on thin layer acrylamide gel. Family studies have shown that the three common phenotypes Fu 1, Fu 2, and Fu 2-1 represent homozygosity or heterozygosity for two autosomal codominat alleles, Fu1 and Fu2.Frequencies of the Fu1 and Fu2 alleles in the New York populations studied were .753 and .247 among whites and .926 and .074 among blacks, respectively.     

Transforming growth factor alpha.

Transforming growth factor alpha (TGFα) is a close relative of epidermal growth factor (EGF), the first polypeptide mitogen discovered in 1962 (Cohen, 1962). TGFα, like EGF, exerts its effect on cells through binding to the EGF-Receptor (EGF-R). Here we review the molecular and cell biology of TGFα before proceeding to describe our own work on signaling molecules induced in response to activation of the EGF-R.     

Metabolic regulation by alpha 1- and alpha 2-adrenoceptors.

The role of alpha-adrenoceptors in the mediation of autonomic function, particularly in the control of the cardiovascular system, is widely known. However, alpha-adrenoceptors are also important in the regulation of a variety of metabolic processes that occur in the body either through direct action or by stimulation of the release of other mediators that control metabolic function. Thus, alpha 2-adrenoceptor activation by circulating or neuronally released catecholamines inhibits the release of insulin from pancreatic islet beta-cells and, by inhibiting this response, alpha 2-adrenoceptor antagonists have been shown to have an antihyperglycemic effect. The alpha-adrenoceptor-mediated regulation of the release of pituitary hormones is indirect, with alpha-adrenoceptors being located on peptidergic neurons in the hypothalamus that secrete releasing hormones into the hypophysial portal system to regulate the secretion of hormones from the anterior pituitary gland. Thus, the increase in cortisol secretion from the adrenal glands following a meal is produced, at least in part, by an alpha 1-adrenoceptor-mediated increase in vasopressin and CRF-41 secretion from neurons on the hypothalamus that stimulate the release of adrenocorticotrophic hormone secretion from the pituitary gland, which subsequently stimulates the synthesis and release of cortisol from the adrenal medulla. In addition to metabolic regulation by alpha 1- and alpha 2-adrenoceptors within the endocrine system, alpha-adrenoceptors are also a component of the system that regulates certain aspects of metabolism within autonomic effector cells, such as the control of smooth muscle cell division and growth during periods of continued alpha-adrenoceptor activation as a result of activation of second messenger systems.     

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.     

The structure of V alpha and J alpha segments in the mouse.

Antigen receptors on most T-cells are heterodimeric glycoproteins, comprised of an alpha chain and a beta chain. These chains are encoded by discontiguous variable (V), diversity (D) and joining (J) gene segments that rearrange to produce a contiguous and functional alpha or beta chain gene. To investigate the size and diversity of the germline repertoire of alpha-chain gene segments, we have characterized and sequenced 20 alpha chain cDNAs. Among these cDNA clones, we have found 4 J alpha and 4 V alpha sequences that have not yet been described. The relationship of these "new" gene segments to those already characterized is discussed.     

Kinetics of folding and unfolding of alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin.

Stopped flow CD (SFCD) kinetic studies of self-assembly of coiled coils of rabbit alpha alpha-tropomyosin and of nonpolymerizable alpha alpha-tropomyosin (NPTm) are reported. The protein was denatured in 6 M urea buffer, then renatured by 10-fold dilution into benign saline buffer. Folding was monitored by SFCD in the backbone region (222 nm). Protein chains are shown to be totally unfolded (and separated in the reduced species) in the initial denaturing medium and fully folded as two-chain coiled coils in the final benign medium. In all cases of folding in benign buffer of totally unfolded chains, two phases were found in the folding process: a fast phase (less than 0.04 s, the SFCD dead time), in which an intermediate state with about 70% of the equilibrium ellipticity forms; followed by a slower, observable phase that completes the folding. The slow phase is first order (k-1 = 1.6 s at 20 degrees C), signifying that chain association for reduced samples occurs in the fast phase. In contrast, folding in benign buffer from an initial state with 70% of the equilibrium ellipticity is all fast, suggesting that the folding intermediate is not an equilibrium species. Cross-linking at Cys-190 increases the helix content of the fast-formed intermediate state to about 85% of the equilibrium value, but leaves the rate constant of the slow phase unchanged. In NPTm, which does not form high aggregates at low ionic strength, the rate of the observable phase is almost independent of ionic strength in the range of approximately 0.15-0.6 M, but is reduced one to two orders of magnitude by further reduction to 0.026 M. In folding from totally unfolded chains, the rate is reduced less than one order of magnitude by changing the final state to about 50% folded. In contrast to folding, unfolding of alpha alpha-tropomyosin from the native state is all fast.     

Triple alpha-genes (alpha alpha alpha anti3.7) in a patient with sickle cell anaemia.

This paper reports the case of a 17-year-old male student from the Jaizan area in south-western Saudi Arabia who had sickle cell anaemia and possessed three alpha-genes on one chromosome (alpha alpha alpha anti3.7) and two on the other. The clinical manifestations were severe, with frequent blood transfusion requirements and frequent episodes of painful crises, severe anaemia and tissue involvement. In comparison with age and sex-matched sickle cell anaemia patients with one alpha-gene deletion (-alpha/alpha alpha), or a normal alpha-gene arrangement (alpha alpha/alpha alpha), a more severe disease presentation was obvious in the propositus. It is suggested that with the surplus alpha-globin chains, more severe haematological and clinical abnormalities occur, these influence the phenotypic expression of sickle cell anaemia. However, more patients with this type of gene arrangement must be studied before a definite conclusion can be reached regarding the influence of excess alpha-globin chains on the presentation of sickle cell anaemia.     

Interferon alpha induces rapid tyrosine phosphorylation of the alpha subunit of its receptor.

The mechanisms of generation of second messengers after binding of interferon alpha (IFN alpha) to its receptor remain unknown. We have studied the phosphorylation of the alpha subunit of the IFN alpha receptor, which is recognized by the monoclonal antibody IFNa receptor 3. Immunoblotting experiments showed that IFN alpha induced rapid tyrosine phosphorylation of the alpha subunit in the IFN alpha-sensitive H-929, U-266, and Daudi cell lines. Immunoprecipitation experiments performed with 32P-labeled cells showed that the alpha subunit is phosphorylated before IFN alpha treatment and that the level of phosphorylation increases after IFN alpha stimulation. Phosphoamino acid analysis confirmed the IFN alpha-induced tyrosine phosphorylation and demonstrated that the base-line phosphorylation corresponded to serine phosphorylation that increased 50% upon IFN alpha treatment. Tyrosine phosphorylation of the alpha subunit was time- and dose-dependent, further demonstrating the specificity of the process. Phosphorylation of the alpha subunit of the receptor occurred rapidly after IFN alpha binding, both at 37 and 4 degrees C. Exposure of the cells to the tyrosine kinase inhibitor genistein blocked the IFN alpha-induced tyrosine phosphorylation of this subunit of the IFN alpha receptor. In contrast H7, a specific protein kinase C inhibitor, and acute and chronic exposure to phorbol esters had no effect on tyrosine phosphorylation, suggesting that protein kinase C does not regulate the tyrosine phosphorylation of the alpha subunit of the IFN alpha receptor. No IFN alpha-induced tyrosine phosphorylation was observed in the IFN alpha-resistant U-937 cell line that expresses a variant IFN alpha receptor. Altogether these data suggest that tyrosine phosphorylation of the alpha subunit may play a role in the signal transduction pathway of IFN alpha.     

Isolation of rat serum alpha 1-microglobulin. Identification of a complex with alpha 1-inhibitor-3, a rat alpha 2-macroglobulin homologue.

Alpha 1-Microglobulin (alpha 1-m), or protein HC, a low molecular weight plasma protein with immunoregulatory properties, was isolated from rat serum by affinity chromatography using Sepharose-coupled monoclonal anti-alpha 1-m antibodies. High molecular weight forms of alpha 1-m were then separated from the low molecular weight alpha 1-m by gel chromatography of the eluted proteins. The apparent Mr (28,000), the charge heterogeneity, the N-linked carbohydrate, and yellow-brown chromophore suggest that the low molecular weight alpha 1-m is the serum counterpart to urinary alpha 1-m, which was purified previously. A high molecular weight complex of alpha 1-m was also isolated by the gel chromatography. It was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. The molecule was bound by antibodies against human alpha 2-macroglobulin, and experiments with antisera against the three alpha-macroglobulin variants in rat serum, alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor-3 (alpha 1I3) suggested that alpha 1I3 was the complex-partner of alpha 1-m. An antiserum raised against high molecular weight alpha 1-m was then used to isolate the complex-partner of alpha 1-m from rat serum with affinity chromatography, and this molecule was positively identified as alpha 1I3 by its physicochemical properties. Gel chromatography of the alpha 1I3.alpha 1-m complex suggested a molecule with an Mr of 266,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, it migrated as three major molecular species with apparent molecular weights of 224,000, 205,000, and 194,000 and several minor species of both higher and lower molecular weights, suggesting a complex subunit structure. alpha 1-m and alpha 1I3 could be detected in all three major species by Western blotting, and NH2-terminal amino acid sequencing suggested a molar ratio of 1:1 of alpha 1-m and alpha 1I3 in all three species. alpha 1I3.alpha 1-m was colorless, did not show light absorbance beyond 300 nm which is typical of low molecular weight alpha 1-m and was electrophoretically homogeneous, suggesting that it lacks the chromophore. Finally, the serum concentrations of the alpha 1I3.alpha 1-m complex and free alpha 1-m were determined as 0.16 and 0.010 g/liter, respectively. Thus, alpha 1I3.alpha 1-m constitutes 1-3% of the total alpha 1I3 in rat serum (w/w) and approximately 60% of the total alpha 1-m.