Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

The determination of intracellular water space by NMR

A new method for the determination of intracellular water space using NMR spectroscopy is described. The method is based on the measurement of 59Co NMR signal intensity of an inert, stable and membrane-impermeable cobalt(III) compound such as Co(CN)3-6 or Co(imidazole)3+6 and the 2H or 1H NMR signal intensities of the freely permeable water. As an example of the method, the variation of the intracellular water space of human erythrocytes as a function of osmolality was measured.     

Structure and expression of the herpes simplex virus type 2 glycoprotein gB gene.

The gene for glycoprotein gB2 of herpes simplex virus type 2 strain 333 was cloned, sequenced, and expressed in mammalian cells. The gB2 protein had an overall nucleotide and amino acid sequence homology of 86% with the cognate gB1 protein. However, of the 125 amino acid substitutions or deletions, only 12.5% were conservative replacements. These differences were clustered within an NH2-terminal region, a central region, and a COOH-terminal region, resulting in domains of near identity broken by small regions of marked divergence. Regions of greatest homology included a 90-amino-acid stretch starting at residue 484 and 39 amino acids spanning residues 835 to 873, which cover a rate-of-entry locus mapped to Ala-552 and a syn locus mapped to Arg-857, respectively, in gB1 by Bzik et al. (D. J. Bzik, B. A. Fox, N. A. DeLuca, and S. Person, Virology 133:301-314, 1984). Pellett et al. (P. E. Pellett, K. G. Kousoulas, L. Pereira, and B. Roizman, J. Virol. 53:243-253, 1985) mapped the mutations in three monoclonal antibody-resistant gB1 mutants between amino acids 273 and 443. These epitopes are included in a region of 98 residues identical between gB1 and gB2. The identity of this protein was verified by placing a truncated gene lacking the 303 carboxyl-terminal amino acids of gB2 into mammalian COS and CHO cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation. This protein will be purified from the stable CHO cell lines and compared with gB1 for immunogenicity and protective efficacy in animal challenge models.     

Human cholinesterase genes localized by hybridization to chromosomes 3 and 16

Summary A cloned human cDNA for cholinesterase (ChE) was used as a probe for in situ hybridization to spread lymphocyte chromosomes to map the structural human CHE genes to distinct chromosomal regions. The recent genetic linkage assignment of the CHE1 locus of the CHE gene to chromosome 3q was confirmed and further refined to 3q21-q26, close to the genes coding for transferrin (TF) and transferrin receptor (TFRC). The CHE1 allele localizes to a 3q region that is commonly mutated and then associated with abnormal megakaryocyte proliferation in acute myelodysplastic anomalies. In view of earlier findings that ChE inhibitors induce megakaryocytopoiesis in culture, this localization may indicate that ChEs are involved in regulating the differentiation of megakaryocytes. A second site for ChEcDNA hybridization was found on chromosome 16q11-q23, demonstrating that the CHE2 locus of the cholinesterase gene, which directs the production of the common C5 variant of serum ChE, also codes for a structural subunit of the enzyme and is localized on the same chromosome with the haptoglobin (HP) gene, both genes being found on the long arm of chromosome 16. The finding of two sites for ChEcDNA hybridization suggests that the two loci coding for human ChEs may include nonidentical sequences responsible for the biochemical differences between ChE variants.     

Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.     

Genetic control of ganglioside biosynthesis in mice

The enzymatic basis for the differences in hepatic ganglioside patterns in the mouse strains C57Bl/6 and Swiss White (SW) was investigated. SW has a “Swiss-type” ganglioside profile, expressing G_M1 ^? and G_D1a ^? in addition to G_M2 ^? as major hepatic gangliosides, whereas C57Bl/6 shows a “G_M2-type” profile, expressing only G_M2 ^? as the major hepatic ganglioside. The enzyme UDP-galactose:G_M2 ganglioside galactosyltransferase (G_M2-GalT), which catalyzes the synthesis of G_M1 ganglioside, showed a four- to fivefold elevation in intact and solubilized liver Golgi membrane fractions of the SW strain compared to C57Bl/6. Crosses between C57Bl/6 and SW produced an F₁ generation with a hepatic ganglioside and enzymatic phenotype intermediate between those of the two parental strains. All three genotypic groups show two forms of the Golgi apparatus enzyme with isoelectric points of 6.5–6.8 and 8.3–9.0. The simplest mode of action of genes which control the enzymatic phenotype that would be consistent with these findings are one or two structural genes or one or two cis-regulatory genes affecting the rate of enzyme synthesis.