Human Beta Subunit Research Articles

Introduction of the α subunit mutation associated with the B1 variant of Tay-Sachs disease into the β subunit produces a β-hexosaminidase B without catalytic activity

Lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) occurs in two major isozyme forms, hexosaminidase A (alpha beta) and hexosaminidase B (beta beta). Although dimer formation is required for enzymatic activity, both subunits contain active sites which share many common substrates. However, the alpha subunit alone confers on hexosaminidase A the specificity for negatively charged substrates, e.g. GM2 ganglioside. Recently, a point mutation, producing a single amino acid substitution in the alpha subunit (Arg178-His), has been found to be associated with the B1 variant phenotype of Tay-Sachs disease (Ohno, K., and Suzuki, K. (1988) J. Neurochem. 50, 316-318). This variant is characterized by normal levels of hexosaminidase A as measured by a common artificial substrate, but an absence of activity toward alpha subunit-specific substrates. However, because of the presence of an active beta subunit in the mutant hexosaminidase A, it has not been possible to determine whether the affected alpha subunit has undergone a change in substrate specificity or become totally inactive. In order to define the full effect of the B1 mutation we have taken advantage of the common evolutionary origin of the genes coding for the alpha and beta subunits. Since the B1 mutation occurs in a region of extended identity between the two subunits, we have duplicated the Arg178-His mutation in a cDNA coding for the human beta subunit (Arg211-His). By expression of the mutant construct in monkey COS cells we have been able to examine the effect of this mutation on beta subunits which are capable of forming stable, active homodimers, an experiment that could not readily be accomplished with heterodimeric hexosaminidase A. Our data show that beta homodimers containing the Arg211-His substitution are formed and are transported into the lysosome in a manner identical to that of normal pro-hexosaminidase B. However, the mutant homodimers are processed at a slower rate and are less stable in the lysozyme. Their most striking feature was a total lack of normal hexosaminidase B activity. We conclude that while the effect of the Arg178-His substitution is not strictly limited to the active site, the severe B1 phenotype results from a totally inactive alpha-subunit in hexosaminidase A.

Cloning and characterization of a cDNA encoding the .beta. subunit of human casein kinase II

Previous studies [Summercorn et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8834-8838; Klarlung & Czech (1988) J. Biol. Chem. 263, 15872-15875] have indicated that Balb/c 3T3 cells and 3T3-L1 adipocytes incubated with insulin show increased casein kinase II activity within minutes, implicating this serine/threonine kinase as an early step in an insulin signaling pathway. We recently reported the isolation of a cDNA encoding an alpha subunit of human casein kinase II [Meisner et al. (1989) Biochemistry 28, 4072-4076] as an initial step toward examining the regulation of this enzyme. We now describe a HepG2 cell casein kinase II beta subunit cDNA of 2.57 kb containing 96 bases of 5' untranslated sequence, 645 bases of open reading frame, and 1832 bases of 3' untranslated sequence with two polyadenylation consensus signal sequences and two poly(A) stretches. The open reading frame of the human beta subunit cDNA was 77% and 87% identical with the Drosophila sequence at the nucleotide and amino acid levels, respectively, and 99% identical with the bovine amino acid sequence. RNA analysis of HepG2 cell RNA utilizing HepG2 beta subunit cDNA fragments as probes revealed one major band migrating at 1.2 kb and two minor bands migrating at 3.0 and 4.2 kb. Results from DNA analysis of HepG2 genomic DNA, consistent with results utilizing Drosophila genomic DNA, suggest the presence of a single gene for the beta subunit of casein kinase II.

Functionally Distinct Agonist and Receptor-binding Regions in Human Chorionic Gonadotropin: Development of a tertiary structure model

Abstract The histidine residues in human chorionic gonadotropin (hCG) were chemically modified using diethyl pyrocarbonate. Derivatives of hCG with an average of 0.5-3.5 histidines modified (maximum of 4 per hCG) had reduced receptor-binding and cell-stimulating activities. Acylation of hCG at progressively lower pH values (conditions in which 1 of the 2 absolutely conserved histidines alpha His-83 is not titratable, whereas alpha His-94 becomes increasingly protonated and resistant to modification) produced hCG derivatives with a greater retention of receptor-binding activity than cell-stimulating activity. The involvement of alpha His-94 as part of the receptor-binding region of the hormone and of alpha His-83 as a putative active site residue was inferred. Proteinaceous protease inhibitors were shown to neutralize the agonist activity of hCG and to reduce the binding of hCG to its receptor and also to specific antisera. It was presumed that an inhibitor-hormone complex was formed which was analogous to the complexing of inhibitor with the of a serine protease. The discovery of primary sequence analogies between hCG and the serine protease chymotrypsin enabled the prediction of hCG structure using the enzyme as a folding template. Solvent-exposed and buried core regions of the peptide chain were delineated using smoothed hydrophobicity profiles in combination with Chou-Fasman secondary structure predictions. Hypervariable hydrophobicity indices between residues 38 and 80 of the human beta subunits reflected different folding arrangements which presumably conferred the individual receptor specificities. When mapped to the putative structure these receptor-determinant loops were adjacent to an area of the alpha subunit analogous to the substrate pocket of serine proteases. Disulfide bond assignments and intersubunit contact regions were identifiable. The proposed tertiary structure for hCG manifests the topographical epitopes defined using monoclonal antibodies and satisfies the currently available data on specific modification and its effects upon hormonal structure and function. This paper is considered to be the first report of a differential effect upon the agonist and receptor binding abilities of a glycoprotein hormone after modification of the proteinaceous, as opposed to the glycosylated, moiety of the molecule.

Rapid radioimmunoassay of circulating chromogranin A: in vitro stability, exploration of the neuroendocrine character of neoplasia, and assessment of the effects of organ failure.

Chromogranin A is a useful probe of neuroendocrine neoplasia in humans. Here we optimize a rapid, sensitive radioimmunoassay modification for detecting chromogranin A in humans and other species. The site of chromogranin A circulation is the acellular plasma; platelets contain no chromogranin A immunoreactivity. The immunoreactivity in plasma is stable to repeated freezing and thawing, prolonged incubation at 37 degrees C, and lyophilization. Venipuncture alone resulted in modest (+ 12%, P less than 0.03) increase in chromogranin A in plasma. Several classic neuroendocrine neoplasia-pheochromocytoma, carcinoid tumor, neuroblastoma, and (vasoactive intestinal polypeptide)oma-produce markedly increased chromogranin A in plasma. By contrast, subjects with malignant melanoma, renal cell carcinoma, and thymoma all had normal values for chromogranin A. Hypersecretion of human choriogonadotropin beta subunit from both malignant (choriocarcinoma) and normal (placenta) syncytiotrophoblast cells was unaccompanied by an increase in chromogranin A, a dissociation compatible with the lack of granular storage and release of syncytiotrophoblastic peptide hormones. Both hepatic and renal failure resulted in increased chromogranin A in plasma, with renal failure leading to concentrations otherwise seen only in neuroendocrine neoplasia. These observations refine the diagnostic specificity of chromogranin A in plasma.

Ontogeny of S-100 protein-positive histiocytes and lymphocytes in the human fetal lymphoreticular system.

In the human lymphoreticular system, the alpha and beta subunits of S-100 protein are found in ordinary monocyte-macrophages and non-phagocytic histiocytes such as Langerhans cells and interdigitating reticulum cells, respectively. The beta subunit is also present in some CD8+ T cells. In the present study, we investigated the ontogeny of these histiocytes and lymphocytes in humans. Yolk sacs and 4 to 21-week fetuses were examined immunohistochemically for the presence of S-100 protein subunits using antisera monospecific to each subunit. S-100 alpha + macrophages were present in the yolk sacs and the hepatic sinusoids of the 4th week embryos prior to bone marrow hematopoiesis. These macrophages later appeared in other lymphoid organs when anlagen of these organs were formed. No S-100 beta + cells were found in the yolk sacs. S-100 beta+ histiocytes were first detected in the hepatic sinusoids of the 5th week embryo, and after the 8th week of gestation, they were distributed in other lymphoid organs. S-100 beta+ lymphocytes were not found in the liver. They were first detected in the thymus at the 12th week of gestation, and were subsequently distributed in other lymphoid organs. These results suggest that S-100 beta+ lymphocytes and histiocytes may belong to different cell lineages, and the former may not be the precursor of the latter.

Human phosvitin/casein kinase type II. Molecular cloning and sequencing of full-length cDNA encoding subunit beta.

Phosvitin/casein kinase type II is an ubiquitous, highly conserved enzyme consisting of subunits alpha, alpha' and beta. Subunit beta, presumably serving regulatory functions, was prepared from human placenta and the amino acid sequence of a protease digestion peptide was determined. The deduced nucleotide sequence was employed for the synthesis of a mixture of 20mers as a hybridization probe to screen a lambda gt10 HeLa cell cDNA library for clones encoding subunit beta. A full-length clone consisting of 1013 bp was isolated and the sequence of both strands determined. The deduced amino acid sequence of the largest open reading frame encodes 215 amino acid residues predicting a maximum Mr of 24925. The nucleotide sequence of the human beta subunit shows a similarity of 75% to that of Drosophila melanogaster and the deduced amino acid sequence a similarity of 88% and 97% to that of D. melanogaster and bovine lung, respectively. No further protein sequence currently known exhibits any notable similarity. Using a 537-bp restriction fragment of the cDNA clone representing 80% of the coding region, a 1-kb subunit-beta transcript was detected by Northern hybridization in total RNA prepared from human epithelial cells and placenta as well as from bovine heart suggesting a single transcript of the beta-subunit gene and also demonstrating high similarity also between the human and bovine nucleotide sequences. The same restriction fragment was used as the probe to indicate in Southern hybridizations that the corresponding genomic DNA contains at least one intron of roughly 2.5 kb in length and presumably is a single copy gene.

Recurrent Germinoma Successfully Treated with Cisplatin

A 23-year-old male with a suprasellar germinoma was initially treated with local irradiation, and the tumor completely disappeared. Three months later, he complained of pain in the left thigh. Myelography revealed multiple spinal metastases at the Th5 to Th8 and Th11 to L5 levels. The tumors were removed through a laminectomy, and the histopathological diagnosis was typical germinoma. Whole spinal irradiation was administered postoperatively. Neurological examination on discharge revealed no abnormality other than hypalgesia over the left L5 segment. Six years later, tumor was found in the lateral ventricles. The human chorionic gonadotropin (HCG) and HCG beta subunit levels were markedly elevated in both serum and cerebrospinal fluid (CSF). Irradiation considerably decreased the size of the tumor but did not completely eradicate it. Subsequent chemotherapy with cisplatin resulted in complete disappearance of the intraventricular tumor, as evidenced by computed tomography (CT). The serum and CSF levels of HCG and HCG beta subunit were well correlated with the tumor size, as shown by CT. The authors emphasize the curative potential of cisplatin in the treatment of recurrent germinoma.

Gastrointestinal Carcinoid Tumours: Histogenetic, Histochemical, Immunohistochemical, Clinical and Therapeutic Aspects

The increased knowledge of the pathobiology of gastrointestinal carcinoid (neuroendocrine) tumours and the improved therapeutic possibilities have brought a demand for more precise diagnosis. Although the carcinoid tumours can often be tentatively recognized in routinely processed microscopic slides, their more accurate identification requires additional diagnostic procedures. General neuroendocrine markers such as the argyrophil reaction of Grimelius and immunohistochemistry with application of antibodies against chromogranin A and of neuron-specific enolase are discriminatory staining methods which are used to reveal the neuroendocrine origin of almost all highly differentiated carcinoid tumours of the gastrointestinal tract. Mid-gut carcinoids, which predominate among these tumours almost unexceptionally contain serotonin. This biogenic amine can be demonstrated by the argentaffin reaction of Masson, serotonin immunoreactively or by formalin-induced fluorescence. The characteristic staining pattern of mid-gut carcinoids is almost invariably preserved in the metastatic deposits and consequently the staining methods for identifying serotonin can also be used on metastases to reveal a primary mid-gut carcinoid. The enterochromaffin-like (ECL) cell carcinoids of the body and fundic area of the stomach often seen in association with pernicious anaemia are argyrophil with the Sevier-Munger silver stain. Other neuroendocrine tumours, viz. antral, duodenal and rectal carcinoids should be studied by a battery of relevant peptide hormone antisera for adequate diagnosis. During the last decade new peptide hormones have been found in circulation in patients with carcinoid tumours, but serotonin and urinary 5-HIAA are still the most important markers for carcinoids of the mid-gut origin. Other clinically useful tumour markers are chromogranin A + B, pancreatic polypeptide, human chorionic gonadotropin alpha and beta subunits. For localizing procedures, angiography is the most reliable investigative method for primary tumours in the gut, whereas CT-scan and ultrasound investigations are good for detection of liver metastases. During the last five years, the therapy for malignant carcinoid tumours has been considerably improved. Chemotherapy has only revealed objective response rates in about 10-30% of the patients giving median survivals from start of therapy of about 10 months. Recently treatment with alpha interferons and the new somatostatin analogue octreotide have given objective responses in 50-75% of patients with malignant mid-gut carcinoid tumours. These patients have now a median survival from start of therapy of 70 months when treated with alpha interferons. In the future new therapies will come into use such as monoclonal antibodies and perhaps also agents blocking different growth factors.

The Follicle-Stimulating Hormone (FSH) Receptor in Testis: Interaction with FSH, Mechanism of Signal Transduction, and Properties of the Purified Receptor1

The follicle-stimulating hormone (FSH) receptor purified from calf bovine testis membranes appears to be an oligomeric glycoprotein, consisting of 4 disulfide-linked monomers of molecular weight about 60,000 each. Polyclonal antibodies to the hormone binding sites of the receptor have been developed. FSH interaction with the receptor seems to involve multiple discrete binding regions, which include amino acids 34-37 and 49-52 of the human FSH beta subunit. The interaction between FSH and the membrane-bound receptor is reversible at low temperatures but becomes increasingly irreversible as the temperature increases. FSH interaction with the soluble receptor is reversible over a wider temperature range. The hydrophobic effect is a significant factor in the initial hormone receptor interaction in each system. FSH bound to membrane receptors on cultured immature rat Sertoli cells is internalized and degraded to the level of amino acids. Current evidence suggests that the membrane receptor may exist as free receptor, and complexed with G-protein. A functional receptor/G-protein/adenylate cyclase complex has been reconstituted in liposomes. The G-protein of testis membranes contains both high and low affinity guanosine triphosphate (GTP) binding sites. Both are capable of modulating FSH receptor binding, whereas only the high affinity sites seem to be required for activation of adenylate cyclase. Although testis membranes contain a phosphatidylinositide hydrolysis system, the latter is not directly influenced by FSH.

Heterogeneity of proteins in commercial preparations of human chorionic gonadotropin (hCG) demonstrated by Western blotting.

Immunoblotting has been utilized to detect the presence of human chorionic gonadotropin (hCG), its alpha and beta subunits, the Tamm-Horsfall protein (uromodulin), immunoglobulins G (IgG) and M (IgM), kappa (K) and lambda (L) chains, and serum albumin in commercially available preparations of hCG intended for human use. Concentrated pregnancy, postpartum, and normal urines were studied as a comparison. Those hCG batches prepared from pooled first-trimester pregnancy urine contained all of the hCG and non-hCG proteins listed above or their fragments, with the single exception of the IgM mu chain. The conflicting literature regarding the immunomodulatory properties of hCG requires reevaluation, since many previous reports of immunologic activity utilized these preparations, containing intact and degraded IgG, K, and L chains, and the Tamm-Horsfall protein, all of which may contribute to an altered immune response. Since hCG can be prepared in high yields, free of these contaminants, these data suggest that patients are being unnecessarily exposed to contaminating substances when receiving parenteral injections of hCG.

Gene structure of the human mitochondrial adenosine triphosphate synthase beta subunit.

The mitochondrial ATP synthase beta subunit is encoded by a nuclear gene and assembled with the other subunits encoded by both mitochondrial and nuclear genes. As the next step in the analysis of the molecular mechanisms coordinating the two genetic systems, the gene for the human beta subunit was cloned, and its structure was determined. The gene contains 10 exons, with the first exon corresponding to the noncoding region and most of the presequence which targets this protein to the mitochondria. Eight Alu repeating sequences including inverted repeats were found in the 5' upstream region and introns. An S1 nuclease protection experiment revealed two initiation sites for the transcription. A typical TATA box was not present at about 30 base pairs upstream from either initiation site. Three CAT boxes (CCAAT) were found between the two initiation sites. In addition, one CAT box was found 41 base pairs upstream from the first initiation site. Two GC boxes (potential Sp1 binding sites) were located in the 5' upstream region, one of them linked to Alu repeating sequences. For determination of the promoter activity, fragments of various length from the 5' upstream region were fused to a chloramphenicol acetyltransferase gene and transfected into cultured cells. This experiment showed the existence of an enhancing, structure(s) for transcription between nucleotide -400 and -1100 in the upstream region.

Structure of platelet glycoprotein IIIa. A common subunit for two different membrane receptors.

The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.

Mapping of human fibronectin receptor beta subunit gene to chromosome 10.

Human-mouse hybrid cells were examined by indirect immunofluorescence with Mab DH12, a monoclonal antibody that recognizes the beta subunit of the human fibronectin receptor. Cells that expressed the antigen at their surface were sorted by FACS and karyotyped. Immunoaffinity chromatography on Mab DH12 was used to confirm the presence of the human antigen. The chromosome assignment was strengthened by isozyme analysis of markers for chromosomes 9 and 10. The results are suggestive for a 10p mapping of this beta subunit of the fibronectin receptor. Since the gene coding for the beta subunit of the VLA proteins was previously assigned to the same chromosome, our result could provide further evidence for the relationship between the beta subunit of the human fibronectin receptor and the VLA protein family.

PstI fragment polymorphism in the gene of the human ATP synthase beta subunit.

ATP synthase (F0F1) is a proton-translocating ATPase which is composed of two moieties, an intrinsic membrane sector (F0) and an extrinsic membrane sector (F1). ATP synthesis is catalyzed by the F1 beta subunit. This paper reports restriction fragment length polymorphism (RFLP) of the human F1 beta subunit gene identified with a cloned cDNA as a probe. Genomic DNAs from 27 unrelated Japanese individuals were analyzed by Southern blot hybridization. RFLP characterized by polymorphic bands at 3.2, 1.7, and 1.5 kilobase-pairs (kbp) was detected inPstI digests of the DNAs, and three genotypes were identified. Of the 27 persons, 16 (59%) were homozygous for the 3.2 kbp band and two (8%) for both the 1.7 and 1.5 kbp bands; the other nine persons (33%) were heterozygous for these band. Memdelian inheritance of thePstI polymorphic bands was shown in 12 members in three generations of one family. The copy number of the F1 beta subunit gene was determined as one.

A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase.

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.

LFA-1 immunodeficiency disease. Definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte function-associated antigen 1 (LFA-1) by complementation in hybrid cells.

Lymphocyte function associated antigen 1 (LFA-1) is a leukocyte cell adhesion protein. We have studied a novel human immunodeficiency disease in which LFA-1 and two other proteins which share the same beta subunit are lacking from the surface of leukocytes. The basis of the inherited defect in cell surface expression of both the alpha and beta subunits of LFA-1 was determined by somatic cell fusion of patient or normal human cells with an LFA-1+ mouse T cell line. Human LFA-1 alpha and beta subunits from normal cells could associate with mouse LFA-1 subunits to form interspecies hybrid alpha beta complexes. Surface expression of the alpha but not the beta subunit of patient cells was rescued by the formation of interspecies complexes. The findings show that the LFA-1 alpha subunit in genetically deficient cells is competent for surface expression in the presence of an appropriate beta subunit, and suggest that the genetic lesion affects the beta subunit. The human LFA-1 alpha and beta subunits were mapped to chromosomes 16 and 21, respectively. The genetic defect is inferred to be on chromosome 21.

Assignment of the gene for the beta subunit of thyroid-stimulating hormone to the short arm of human chromosome 1.

The chromosomal locations of the genes for the beta subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).

Characterization of cleavage products in selected human lutropin preparations. A protease-sensitive site in human lutropin beta subunit.

Low molecular weight fragments derived from the beta subunit of human lutropin have been frequently observed. These fragments are detected by polyacrylamide gel electrophoresis in sodium dodecyl sulfate following reduction of the disulfide bonds. A sample of human lutropin was identified that had a major portion of its beta subunit showing this proteolytic nick. Over 83% of the subunit was nicked based on reduction, carboxymethylation, and isolation of the low molecular weight fragments. This preparation had 53% of the activity of an intact human lutropin (radioligand assay). The proteolytic nick in the subunit was shown by N-terminal sequencing of the C-terminal fragments to be derived from three clips in a hexapeptide region (residues 44-49) characterized by hydrophobic alkyl side chains. Specific clips were on the amino side of Leu-45 (8%), Val-48 (45%) and Leu-49 (47%). Thus the proteolytic activity, presumably derived from the pituitary during processing, has a substrate specificity reminiscent of the bacterial protease, thermolysin.

A spectrin-like protein from mouse brain membranes: phosphorylation of the 235,000-dalton subunit.

A mouse brain spectrin-like protein, which was an immunoreactive analogue of erythrocyte spectrin, has been isolated from demyelinated membranes. This spectrin analogue was a 10.5 S, 972,000 molecular weight (Mr) (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) Mr. We demonstrated that in vivo only the 235,000 Mr beta subunit of the mouse brain spectrin-like protein was phosphorylated, which was an analogous situation to mouse erythrocyte spectrin in which only the 220,000 Mr beta subunit was phosphorylated. Incubation of isolated membrane fractions with [gamma-32P]ATP +/- adenosine 3',5'-cyclic monophosphate (cAMP) indicated that mouse brain spectrin-like protein, mouse erythrocyte spectrin, and human erythrocyte spectrin's beta subunits were all phosphorylated in vitro by membrane-associated cAMP-independent protein kinases.

Molecular cloning and characterization of a cDNA for the beta subunit of human alcohol dehydrogenase.

Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related alpha, beta, and gamma polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the beta subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing clone, pADH12, which contained an 1100-base-pair cDNA insert, was subjected to DNA sequence analysis. The sequence indicated that the cDNA encoded information for the carboxyl-terminal 91 amino acids of a class I ADH and a 3' untranslated region of 593 nucleotides. Comparisons with the carboxyl terminus of the human ADH beta subunit indicated that the cDNA encoded the beta polypeptide. This probe may facilitate genetic studies of various human alcohol-related syndromes, as well as enable basic molecular studies on human ADH gene expression.