Rethinking the role of phosducin: light-regulated binding of phosducin to 14-3-3 in rod inner segments.

Phosducin (Pd), a small protein found abundantly in photoreceptors, is widely assumed to regulate light sensitivity in the rod outer segment through interaction with the heterotrimeric G protein transducin. But, based on histochemistry and Western blot analysis, Pd is found almost entirely in the inner segment in both light and dark, most abundantly near the rod synapse. We report a second small protein, 14-3-3, in the rod with a similar distribution. By immunoprecipitation, phospho-Pd is found to interact with 14-3-3 in material from dark-adapted retina, and this interaction is markedly diminished by light, which dephosphorylates Pd. Conversely, unphosphorylated Pd binds to inner segment G protein(s) in the light. From these results and reported functions of 14-3-3, we have constructed a hypothesis for the regulation of light sensitivity at the level of rod synapse. By dissociating the Pd/14-3-3 complex, light enables both proteins to function in this role.

C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.

Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains.

The structure of an N-terminal fragment of CD4 has been determined to 2.4 A resolution. It has two tightly abutting domains connected by a continuous beta strand. Both have the immunoglobulin fold, but domain 2 has a truncated beta barrel and a non-standard disulphide bond. The binding sites for monoclonal antibodies, class II major histocompatibility complex molecules, and human immunodeficiency virus gp120 can be mapped on the molecular surface.

A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes.

In a T cell antigen receptor complex (TCR), the clonotypic disulfide-linked Ti heterodimer is noncovalently associated with the invariant CD3 polypeptides. The latter are composed of three monomeric subunits (gamma, delta, epsilon) and either a disulfide-linked homodimer (zeta zeta) or a disulfide-linked heterodimer (zeta eta). The exact stoichiometry of the Ti-CD3 subunits in a given complex is still largely unknown. Here, we report the presence of a CD3 epsilon dimer in a fraction of the TCR. When TCRs from both human and murine T lymphocytes were immunoprecipitated with monoclonal antibodies against either CD3 epsilon or Ti, a 40-kDa disulfide-linked dimer was coprecipitated with the other TCR subunits from digitonin lysates. Amino acid sequence analysis of peptides obtained by in situ CNBr cleavage of the 20-kDa product blotted to polyvinyl difluoride membranes from reducing/nonreducing two-dimensional gels identified human CD3 epsilon. Assuming this CD3 epsilon to derive from a homodimer, then either some TCRs contain more than one CD3 epsilon chain or several TCRs are covalently associated with one another via their CD3 epsilon subunits. Although it has been suggested that a putative TCR association with CD2 exists under similar conditions to those utilized to detect CD3 epsilon dimers, the CD2 molecule was not coimmunoprecipitated with the TCR by any of a series of anti-CD3 epsilon monoclonal antibodies. In conjunction with the fact that CD2 and the TCR do not colocalize during conjugate formation between T cells and antigen-presenting cells (Koyasu, S., Lawton, T., Novick, D., Recny, M. A., Siliciano, R. F., Wallner, B. P., and Reinherz, E. L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2603-2607), we conclude that CD2 and the TCR are not physically associated on the T cell surface.

Molecular cloning of the CD3 eta subunit identifies a CD3 zeta-related product in thymus-derived cells.

The CD3 eta subunit of the T-cell antigen receptor forms a heterodimeric structure with the CD3 zeta subunit in thymus-derived lymphoid cells and is apparently involved in signal transduction through the receptor. Here we report the primary structure of murine CD3 eta as deduced from protein microsequencing and cDNA cloning. The mature protein is divided into three domains: a 9-amino acid extracellular segment, a 21-amino acid transmembrane segment including a negatively charged residue characteristic of CD3 subunits, and a 155-amino acid cytoplasmic tail. The NH2-terminal sequences of CD3 eta and CD3 zeta are identical through amino acid 122 of each mature protein but then diverge in the remainder of their respective COOH-terminal regions, consistent with alternatively spliced products of a common gene. The cytoplasmic domain of CD3 eta is 42 amino acids larger than that of CD3 zeta but lacks one of six potential tyrosine phosphorylation sites as well as a putative nucleotide binding site previously identified in CD3 zeta. These structural features presumably account for the difference between CD3 eta and CD3 zeta function and are consistent with the notion that CD3 eta may be an important component of a T-cell receptor isoform(s) during thymic development.

A comparative study of bark lectins from three elderberry (Sambucus) species.

Three elderberry lectins isolated from the bark of three different species of the genus Sambucus which are native to Europe (S. nigra), North America (S. canadensis), and Japan (S. sieboldiana) were studied comparatively with regard to their carbohydrate binding properties and some structural features. All three lectins contained two identical carbohydrate binding sites per molecule and showed a very high specificity for the Neu5Ac(alpha 2-6)-Gal/GalNAc sequence. However, relative affinities for various oligosaccharides were significantly different among them, suggesting differences in the detailed structure of the carbohydrate binding sites of these lectins. The three lectins were immunologically related, but not identical, and all were composed of hydrophobic and hydrophilic subunit regions, although the molecular sizes of these subunits were slightly different among the three lectins. N-terminal sequence analysis of the subunits of these lectins suggested that they have a very similar structure in this region but also indicated the occurrence of N-terminal processing such as the deletion of several amino acid residues at the N-termini for both hydrophobic and hydrophilic subunits of all three lectins. Tryptic peptide mapping of the three lectins showed a similar pattern for all of them but also showed the presence of some unique peptides for each lectin.

The activator protein for glucosylceramide beta-glucosidase from guinea pig liver. Improved isolation method and complete amino acid sequence.

beta-Glucosidase activator (SAP-2) is a family of heat-stable, acidic glycoproteins which stimulate enzymatic hydrolysis of glucosylceramide. In this study, we improved the purification method and found that SAP-2 is highly heterogeneous. A hot water extract of frozen guinea pig liver was fractionated by ammonium sulfate sedimentation, then chromatographed with DEAE-Sephacel, Sephadex G-75, and concanavalin A-Sepharose. A fraction binding to concanavalin A-Sepharose was purified further with a C4 high performance liquid chromatography reverse phase column. This yielded several peaks, the main one of which was studied. The specific activity of the purified SAP-2 was 35 units/micrograms (1 unit produces 50% stimulation of a basal glucosidase preparation). N-terminal amino acid sequencing showed that this preparation is a mixture of polypeptides differing in the presence or absence of one or two of the end amino acids. The complete amino acid sequence of the 81 residues in SAP-2 has been determined. Comparison of the sequence of guinea pig SAP-2 with the sequence of human sphingomyelinase activator revealed 58% homology and quite similar hydropathy profiles. Both proteins possess a highly hydrophilic region around Asn-22, which is glycosylated, and 6 cysteine residues, in oxidized form, located in the same positions. Comparison with the published nucleotide sequence for the precursor form of the human activator protein for sulfatide sulfatase (SAP-1) suggested that this activator also has a possibly glycosylated Asn and 6 Cys residues at similar positions, although the remainder of the molecule is somewhat different. Examination of another region of the precursor's nucleotide sequence, assuming a few changes in the identifications, revealed the presence of the sphingomyelinase activator. It appears that two or more activators are derived from a single precursor protein. Marked homologies were seen also with a lung surfactant protein and a sulfated glycoprotein from Sertoli cells.

Purification, physicochemical, and kinetic properties of liver acetyl-CoA:arylamine N-acetyltransferase from rapid acetylator rabbits.

Cytosolic liver acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from homozygous rapid acetylator rabbits (strain III/J) was purified to homogeneity as judged by gel filtration sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and isoelectrofocusing. The isoelectric point was estimated to be 5.2. The molecular weight was determined to be 33,500 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and 33,000 by Sephacryl S-200 gel filtration. The amino acid composition is reported and 16 tryptic peptides were sequenced by Edmann degradation, including a peptide from which a very specific oligonucleotide probe can be synthesized. The enzyme contained neither amino sugars nor cofactors. A broad pH optimum from pH 5.9 to 8.6 was observed. N-Acetyltransferase activity showed a strong dependency on the salt concentration. From the influence of the basicity of the acceptor amine on the maximum velocity, it was concluded that the formation of the covalent acetyl-enzyme intermediate is the rate-limiting step in the N-acetyltransferase-catalyzed acetylation of amines. The covalent intermediate reacts, then, in a fast step with the acceptor amine, when using aniline derivatives with pKa values ranging from 5.65 to 1.74. However, with the weakly basic 4-nitroaniline, the acetyltransfer from the catalytic intermediate to the amine seems to be rate-limiting. A structure-activity study of 30 aniline derivatives that differ in hydrophobicity, position, size, charge, and number of substituents showed that some ortho-substituted derivatives were not acetylated.

Primary structure of an analog of crustacean pigment-dispersing hormone from the lubber grasshopper Romalea microptera.

An octadecapeptide capable of inducing pigment dispersion in the chromatophores of the fiddler crab Uca pugilator has been isolated from lyophilized heads of the lubber grasshopper Romalea microptera. This pigment-dispersing factor (PDF) was purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase high performance liquid chromatography. Automated gas-phase sequencing, followed by the identification of the carboxyl-terminal amide, established the primary structure of this PDF as Asn-Ser-Glu-Ile-Ile-Asn-Ser-Leu-Leu-Gly-Leu-Pro-Lys-Leu-Leu-Asn-Asp-Ala- NH2. This structure was confirmed by chemical synthesis and by demonstrating that the synthetic and native PDF displayed identical chromatographic behavior and biological activity. The Romalea PDF is structurally related to the crustacean pigment-dispersing hormones (PDHs), which are also octadecapeptides. The sequence of grasshopper PDF shows 78% homology with beta-PDH (from the crabs U. pugilator and Cancer magister) and 50% homology with alpha-PDH (from the prawn Pandalus borealis). This study provides the first direct chemical evidence for the structural relatedness of insect PDF to the crustacean PDHs, thus identifying them as an authentic family of arthropod peptides.

Isolation of a cDNA coding for human galactosyltransferase.

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a lambda gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 X 10(6) recombinant lambda gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.

Isolation of galactosyltransferase from human milk and the determination of its N-terminal amino acid sequence.

Galactosyltransferase (EC 2.4.1.22), purified to homogeneity from human milk by affinity chromatography, had an apparent molecular weight of 53,000 as determined by denaturing polyacrylamide gel electrophoresis. Subtraction of the estimated contribution of the oligosaccharide portion of the molecule leaves a Mr of 47,000. An N-terminal amino acid sequence analysis of the isolated protein revealed a sequence similar to that found near the 5' end of a cDNA clone isolated by Shaper et al, which encodes a 35,000 molecular weight protein. Either the molecular weight of galactosyltransferase, has been overestimated, or a discrepancy exists between the actual molecular weight of galactosyltransferase and that predicted by the bovine cDNA clone isolated by Shaper et al.

Characterization of a pigment-dispersing hormone in eyestalks of the fiddler crab Uca pugilator.

A pigment-dispersing hormone (PDH) from eyestalks of the fiddler crab Uca pugilator has been purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase liquid chromatography. Based on automated gas-phase sequencing and subsequent identification of carboxyl-terminal amide, we have assigned the primary structure of this peptide as Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Asp-Ala-NH (2). We have confirmed the sequence by synthesizing this peptide and demonstrating that the synthetic PDH and the native PDH display identical chromatographic behavior and biological activity. This hormone is a member of a family of invertebrate neuropeptides that includes a light-adapting/pigment-dispersing octadecapeptide hormone from the prawn Pandalus borealis. In assays for melanophore pigment dispersion in destalked fiddler crabs, Uca PDH was 21-fold more potent than Pandalus PDH. These two hormones share a hexapeptide core sequence (residues 5-10: -Ile-Asn-Ser-Ile-Leu-Gly-) as well as the amino- and carboxyl-terminal residues but differ at positions 3, 4, 11, 13, 16, and 17. These results point to speciesrelated or group-specific structural differences among crustacean PDHs.

On the amino acid sequence of cytochrome P-450 isozyme 4 from rabbit liver microsomes.

Isozyme 4 of rabbit liver microsomal cytochrome P-450 was shown earlier in this laboratory to contain multiple NH2-terminal residues, whereas isozymes 2, 3a, 3b, and 3c have single, unique NH2-terminal sequences. Similar results were obtained with isozyme 4 obtained from animals that were untreated, treated with phenobarbital (which does not induce this isozyme), or induced with beta-naphthoflavone or isosafrole. With the use of selective chemical blocking at seryl or at nonprolyl residues, the complexity of the NH2-terminal sequence has now been shown to be due to the presence of three forms of the cytochrome differing only in the absence of the first or the first two residues: NH2-Ala-Met-Ser-Pro-Ala-Ala-Pro-, NH2-Met-Ser-Pro-Ala-Ala-Pro-, and NH2-Ser-Pro-Ala-Ala-Pro-. These forms may result from variable biological processing. Peptides containing the seven cysteine residues were sequenced and compared with similar peptides reported for other P-450 cytochromes; homology was extensive with respect to two of the cysteine regions in isozyme 4, and a third cysteine region showed partial identity. The sequence of peptides representing about two-thirds of the amino acids in isosafrole-induced cytochrome P-450 isozyme 4 was determined. Comparison with phenobarbital-induced rabbit cytochrome P-450 isozyme 2 indicated about 25% homology. In contrast, comparison of isozyme 4 with rat cytochrome P-450d, which is also induced by isosafrole and for which the sequence has recently been deduced from cDNA [Kawajiri, K., Gotoh, O., Sogawa, K., Tagashira, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. USA 81, 1649-1653], showed about 70% homology.

Respiratory proteins from the extremely thermophilic aerobic bacterium, Thermus thermophilus. Purification procedures for cytochromes c552, c555,549, and c1aa3 and chemical evidence for a single subunit cytochrome aa3.

We have developed a chemically defined, minimal growth medium for Thermus thermophilus which is suitable for nutritional studies, isotopic enrichment, and genetic manipulation of the organism. Reliable procedures are described for the large scale purification of cytochrome c552 from the periplasm and for cytochrome c555,549 and cytochrome c1 aa3 from the plasma membrane. In contrast to a previous report (Fee, J. A., Choc, M. G., Findling, K. L., Lorence, R., and Yoshida, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 147-151) which suggested a molecular weight near 200,000, the cytochrome c1aa3 complex was shown by protein and amino acid analyses to have Mr approximately 93,000. Sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography, combined with amino acid analyses, revealed the presence of only two proteins in a 1:1 ratio: C-protein has Mr approximately 33,000, binds heme C, and is thought to correspond to cytochrome c1. A-protein has Mr approximately 55,000 and is thought to bind the four redox components (2 heme A and 2 Cu) of cytochrome aa3.

Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands.

We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.

Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes.

The complete amino acid sequence of phenobarbital-induced isozyme 2 of rabbit liver microsomal cytochrome P-450 (P-450LM2) is presented. The polypeptide consists of 491 residues with a calculated Mr of 55,755. The rabbit isozyme is 77% identical to the corresponding rat cytochrome, P-450b, as deduced from cDNA, with 96% of the hydrophobic, 88% of the anionic, and 83% of the cationic positions conserved. The secondary structure of isozyme 2 was predicted and a model was developed for the membrane topology of this cytochrome. Of the two highly conserved cysteinyl peptides in P-450LM2, P-450b, and bacterial P-450cam, we favor, on the basis of our model, the one nearer the NH2 terminus (Cys-152 in P-450LM2) as the source of the thiolate ligand to the heme iron atom. The recently reported sequence of the apparently identical protein [Heinemann, F. S. & Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201] has two fewer residues and differs in 14 other amino acid assignments.