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Received September 8, 1997; accepted January 5, 1998 The structural gene encoding the Rieske iron-sulfur protein from Thermus thermophilus HB8 has been cloned and sequenced. The gene encodes a protein of 209 amino acids that begins with a hydrophilic N-terminus followed by a stretch of 21 hydrophobic amino acids that could serve as a transmembrane helix. The remainder of the protein has a hydrophobicity pattern typical of a water-soluble protein. A phylogenetic analysis of 26 Rieske proteins that are part of bc\ or b6f complexes shows that they fall into three major groups: eubacterial and mitochondrial, cyanobacterial and plastid, and five highly divergent outliers, including that of Thermus. Although the overall homology with other Rieske proteins is very low, the C-terminal half of the Thermus protein contains the signature sequence CTHLGC-(13X)-CPCH that most likely provides the ligands of the [2Fe-2S] cluster. It is proposed that this region of the protein represents a small domain that folds independently and that the encoding DNA sequence may have been transferred during evolution to several unrelated genes to provide the cluster attachment site to proteins of different origin. The role of individual residues in this domain of the Thermus protein is discussed vis-a-vis the three-dimensional structure of the bovine protein (Iwata et al., 1996 Structure 4, 567-579).

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.

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.

A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.

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.

We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.

Publisher Summary Protein sequence determination is carried out most frequently by the Edman degradation. This chapter describes and examines both the kinetics of the O-to-N acyl shift and the catalysis of thiocarbamylation to determine optimal conditions. The results show that O-to-N shift can be prevented by: using anhydrous coupling; excluding amines and/or by lowering the pH of the coupling reaction to pH 5. The chapter discusses that the major contributor to background generation is the result of an N-to-O shift at Ser/Thr hydroxyl groups. A blocking group on Ser/Thr can prevent N-to-O shifting, but must be stable and not O-to-N shift when Ser/Thr become N-terminal. The chapter reveals that thiocarbamylation does not require tertiary amines, and that efficient coupling, around neutral pH, can be carried out in the presence of tetraethylammonium sulfate and methylsulfoxide, with model compounds the O-to-N shift is not promoted. It may be expected that a combination of non-tertiary amine catalysis and acylating reagent with bulky side chain suppresses O-to-N shift, while maintaining high repetitive efficiency.

Publisher Summary The ABRF studies to date have examined the recovery, accuracy, and precision of amino acid analysis using either synthetic peptides, purified proteins, or a protein hydrolysate. These studies concentrate on the sensitivity of analysis with hydrolyse and analyses being performed in the 0.1–5 μg range. The accuracy of analyses has generally been in the 80–90% range. This chapter discusses an attempt at investigating the quality of analyses under conditions where laboratories might feel more comfortable and where ample supply of sample was available. Three general aspects were explored. The first two were a realistic assessment of the accuracy of absolute quantitation and of the accuracy of relative yields of residues obtainable at present. The third aspect was the analysis of the problem amino acids tryptophan and cystine/cysteine.

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.

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.

The entire amino acid sequence of hypoxanthine-guanine phosphoribosyltransferase from human erythrocytes has been defined. Peptide fragments formed by cleavage at arginine, glutamic acid, and methionine residues were analyzed by Edman degradation or digestion with carboxypeptidase. The complete primary structure of human hypoxanthine-guanine phosphoribosyltransferase was established by sequence analysis of 17 peptide fragments, 15 of which were purified by reverse-phase high pressure liquid chromatography. The enzyme is 217 residues long with a molecular weight equal to 24,470. Mass spectroscopy indicated that the NH2-terminal alanine is acetylated.

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.

Earlier findings in several laboratories have suggested that the various forms of cytochrome P-450 within a given species or from different species are not closely related. However, our present studies on the primary structure of the phenobarbital-inducible cytochrome from rabbit liver microsomes (P-450LM2) show that this protein is about 80% identical to the corresponding rat protein (P-450b), the sequence of which was recently deduced by others. For example, of the first 70 residues of the NH2-terminal sequences, 52 are identical, with most differences structurally conservative and attributable to point mutations. No evidence was found for heterogeneity in the region of the rabbit cytochrome corresponding to the variable region proposed for the rat cytochrome. A comparison of the known primary structure of P-450LM2 with that of P-450b and that of the bacterial camphor-hydroxylating cytochrome (P-450cam) as recently reported by others reveals only one highly conserved cysteine-containing peptide present in approximately the same position in each cytochrome. This peptide in the rabbit enzyme, -Arg-Ile-Gln-Glu-Glu-Ala-Arg-Cys147-Leu-Val-Glu-Glu-Leu-Arg-, and the corresponding peptides containing Cys152 in P-450b and Cys134 in P-450cam may serve an essential function such as providing the axial thiolate ligand to the heme iron atom.

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.

Modifications of the classical Edman cycle permitted the complete sequential degradation, without prior derivatization, of all small and medium-sized peptides with a free N-terminus. Sequences of up to 40 residues appeared possible with most long peptides. Quantities of 5–50 nmoles were optimal for the apparatus described. Cycling time was 50–90 min. Degradation was monitored and derivatives identified with a fast and sensitive thin-layer technique.

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.

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.

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.

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 Mossbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mossbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mossbauer 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 Mossbauer 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 Mossbauer 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.

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.

Hematologic evaluation of a Nigerian obstetrical patient disclosed the presence of sickle-cell trait as well as evidence of a hemoglobin alpha- chain abnormality. Hemoglobins containing the variant alpha-chain were isolated by DEAE-cellulose column chromatography, and analysis of the purified alpha-chain demonstrated a ser replaced by cys substitution at alpha-81. The abnormal alpha-chain represented approximately 45% of the total, and hemoglobins containing this alpha-chain appeared to have normal stability and functional properties. In addition to the abnormal hemoglobins that were identified in this patient, she also was found to have persistent microcytosis in the absence of iron deficiency, and the percentage of HbS in her erythrocytes was less than that usually present in individuals with sickle cell trait. These findings, together with a reduced alpha/beta globin synthesis ratio from her peripheral blood reticulocytes, indicated that the presence of alpha-thalassemia trait. Hematologic findings from members of the patients's family suggest that an alpha-thalassemia gene may be linked to that of the structurally abnormal alpha-chain.

Two-dimensional peptide mapping has been very effective in the characterization of protein digests, particularly for the detection of small structural differences between homologous proteins. The classical thin-layer strategy, which exploits differences in charge and hydrophobicity, has been realized as a method based on reversed-phase high-performance liquid chromatography. An initial fractionation at pH 7.2 with 100 m M potassium phosphate, followed by chromatography with 0.1% trifluoroacetic acid, has been applied to chymotryptic digests of cytochromes c . The use of UV-transparent and (in the final stage) volatile solvents allows detection and rapid recovery of nanomole amounts of peptides suitable for sequence determination. As an example of the application of this method we report the comparison of two variants of cytochrome c from the butterfly, Pieris brassicae , one being the wild type and the other a spontaneous mutant isolated from a laboratory colony. The single residue difference was easily detected and identified.

Hematologic evaluation of a Nigerian obstetrical patient disclosed the presence of sickle-cell trait as well as evidence of a hemoglobin alpha- chain abnormality. Hemoglobins containing the variant alpha-chain were isolated by DEAE-cellulose column chromatography, and analysis of the purified alpha-chain demonstrated a ser replaced by cys substitution at alpha-81. The abnormal alpha-chain represented approximately 45% of the total, and hemoglobins containing this alpha-chain appeared to have normal stability and functional properties. In addition to the abnormal hemoglobins that were identified in this patient, she also was found to have persistent microcytosis in the absence of iron deficiency, and the percentage of HbS in her erythrocytes was less than that usually present in individuals with sickle cell trait. These findings, together with a reduced alpha/beta globin synthesis ratio from her peripheral blood reticulocytes, indicated that the presence of alpha-thalassemia trait. Hematologic findings from members of the patients's family suggest that an alpha-thalassemia gene may be linked to that of the structurally abnormal alpha-chain.

We have investigated the molecular basis for a deficiency of the enzyme hypoxanthine (guanine) phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) in a patient with a severe form of gout. We reported in previous studies the isolation of a unique structural variant of HPRT from this patient's erythrocytes and cultured lymphoblasts. This enzyme variant, which is called HPRTLondon, is characterized by a decreased concentration of HPRT protein in erythrocytes and lymphoblasts, a normal Vmax, a 5-fold increased Km for hypoxanthine, a normal isoelectric point, and an apparently smaller subunit molecular weight. Comparative peptide mapping experiments revealed a single abnormal tryptic peptide in HPRTLondon. Edman degradation of the aberrant peptide from HPRTLondon identified a serine-to-leucine amino acid substitution at position 109. This substitution can be explained by a single nucleotide change in the codon for serine-109 (UCA leads to UUA). Thus a mutation at the HPRT locus has now been defined at the molecular level.

Free and derivatized aglycones and parent ascarosides of Ascaris lumbricoides and A. columnaris (Nematoda) were isolated by thin-layer and gas chromatography and analyzed by mass spectrometry and polarimetry. Results for monol aglycones confirmed previous chain-length assignments and the location of the l -hydroxyl group at C-2. The major monols of even carbon number possessed a methyl branch on the penultimate carbon (ω − 1). Diol alycones were entirely unbranched homologues, mainly 31 and 33 carbons long, and had hydroxyl groups on C-2 and C-(ω − 1). These aglycones were truly symmetrical, for they were optically inactive with the center bearing the glycone in diol ascaroside (C-2), being mostly or entirely the l configuration. Three major features of ascaroside structure—chain length composition of the aglycones, molecular weight of the unusual glycone (a dideoxyhexose), the kind and position of acyl groups—were clearly discernible in the spectra of intact ascarosides.

1. 1. Free and esterified ascarosides were identified in the ascarids Ascaris columnaris, Ascaridia galli and Toxocara cati , and the oxyurid Passarulus sp. through comparison with authentic ascarosides from Ascaris lumbricoides . 2. 2. The proportions of monol ascaroside (MS), diol ascaroside (DS) and diol diascaroside (DSS) were roughly the same in all species. 3. 3. Aglycones averaged 29.2–30.4 carbons in MS, 31.2–34.4 in DS and 31.7–35.3 in DSS. A variable percentage of ascarid monol aglycones were branched. 4. 4. Ascaroside esters of all species were primarily acetates and had identical isomeric form. Percentages of propionate and unesterified hydroxyl groups in ovarian ascarosides, and of volatile acids in triglycerides and waxes, increased with increasing size of the parasite.

Glycerides (65%), ascaroside esters (33%), waxes (1.5%) and sterols (0.26%) accounted for essentially all the neutral lipids inA. lumbricoides ovaries. Nineteen per cent by weight of the triglycerides contained only long chain fatty acids. Nearly all the remaining triglycerides contained 1 mol volatile acid. Mono- and diglycerides, free fatty acids and triglycerides containing 2 mol volatile acids were present in very small amounts. Mole percentages of glyceride volatile acids were α-methylavaleric (70), α-methylbutyric (23),n-valeric (ca. 7), and traces of acetic, propionic, isobutyric andn-butyric. Mole percentages of the alcoholic component of the waxes were 96% 1-octadecanol, and 4% of its 16, 17 and 19 carbon homologs. The acid components were α-methylbutyric (75 mol%), acetic (16 mol%), propionic andn-valeric (each 4 mol%), and traces ofn-butyric and α-methylvaleric. Sterols (42 wt% cholesterol, 30% cholestanol, 9.5% campestanol, 8.2% stigmastanol, 6.6% β-sitosterol, and 3.8% campesterol) were essentially the same as those found in the whole worm, except that no esterified sterols were present.

Until recently the rate-limiting step in protein structural determination was the purification of peptides, so sequencing efforts focused on long degradations of large fragments. With the advent of HPLC methods the sequencer has been confronted with a host of suitable material mostly of small to moderate size. Improvements in procedure for automatic instruments like the spinning cup have extended their applicability, range and sensitivity, but have only slightly increased their speed: one day’s worth of HPLC runs can easily produce enough peptides to occupy an automatic sequenator for half a year.

Publisher Summary This chapter considers the properties and merits of the manual mode and its chemical and mechanical implementation. Manual methods have all the options and capabilities of automatic methods plus others that are not yet automated, such as multiple sample processing. Besides being flexible, manual methods are cheap, especially with regard to capital outlay. Any sequencing strategy can be realized in either an automatic or manual mode. A comparison of the merits of each mode suggests the continued application of manual methods to (1) limited sequencing projects with small budgets; (2) large numbers of smaller-sized peptides (to about 20 residues); (3) cases requiring special handling; (4) the characterization of NH 2 -terminal regions for identification; and (5) screening prior to attempts at complete, automatic degradation. Successful sequencing depends more on fundamental understanding of Edman chemistry than on hardware. The factors considered in this chapter are relevant to all approaches, but are most specifically related to liquid-partition with direct identification of the derivatized products.

Summary Lysosome solubilized pig liver cytochrome b5 reductase has the following twenty residues as NH2-terminal sequence: Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp-Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp. The fragment is predicted to exist largely in the random conformation with 2 β-bends at residues 9–12 and 14–17. The reductase fragment appears clean enough for complete sequence investigation and is very similar to cathepsin D-solubilized rabbit liver cytochrome b5 reductase.

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 ~gtlO cDNA library. Two hybridizationpositive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 x 106 recombinant ~gtlO phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransf~rase coding sequence. The remainder of the sequence corresponded to the 3 -region of the mRNA downstream from the termination codon. ® 1986 Academic Press, Inc.

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.

Automatic spinning-cup sequenators are often incapable of approaching the carboxy terminus of a peptide because of the mechanical loss of material (washout). We report here an adjunct, polyquarternary amines, specifically Polybrene, that greatly reduces or eliminates this problem.

A complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in man leads to the development of the Lesch-Nyhan syndrome (1) whereas a partial deficiency of the same enzyme leads to a severe form of gout (2). In an attempt to understand the molecular basis of these deficiency states, we have focused our recent studies on the structural and functional properties of HPRT from normal and enzyme-deficient patients.

Methods for obtaining sequence information at the C-terminus of a protein, ideally as efficiently and conveniently as at the N-terminus using the Edman degradation, have been of perennial interest to protein chemists (see (2) for review). And now molecular geneticists would like information sufficient for construction of a probe, especially as the N-terminus is all too frequently blocked, and for verifying reading frames. The most popular current method for direct C-terminal sequencing is by carboxypeptidase, usually Y (3); besides the problems of phasing, widely varying residue- or sequence-specific rates, and unpredictable digestion conditions, the method apparently predicts a structure only rarely resembling the true sequence (in only 2 of 12 cases for cytochromes P-450 (4)). Of many chemical methods, only the 60-yr old thiocyanate degradation developed to practicality by Stark (5) has attained appreciable success, but even with repeated improvements (most recently, (6)) it seems not to have contributed any new sequence information. An alternative is the indirect “back door” approach of isolating from a digest of a protein that peptide(s) representing the C-terminal region, followed by Edman sequencing. This requires tagging the C-terminus before digestion and a convenient way of picking the tagged peptide from among the many others. For instance, all protein carboxyl groups, side-chain and C-terminal, could be reacted with an amine. Digestion restores one carboxyl group to each peptide except for the C-terminal one, thus permitting discrimination on the basis of this group (7), for instance by anion exchange chromatography; the unbound C-terminal peptide can be transferred directly to a reversed phase column for final purification. We have used an amidation procedure for several years that increases solubility and unfolds proteins to enzymatic digestion, and which gives peptides a definitive end-of-sequence signal (1,8), so our isolation of the C-terminal peptide extends this procedure.

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LMI were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LMI was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02-o. 1 M hexafluoroacetone/ NH3, pH 7.2 for highly acidic peptides; and (2) 6 M formic acid/O.13 M trimethylamine, pH 1.5, vs 4 M formic acid/O.09 M trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterilication of serine and threonine residues and appear to be reversible by aminoethanol treatment.

One of the delights of working with proteins, as opposed to nucleic acids, is their great variation in properties. But for the protein sequencer concerned only with covalent structure, this is also a problem. For example, a protein which is acidic or easily precipitated by organic solvents may not elute from a reversed-phase HPLC column under the standard conditions of dilute acid and acetonitrile (MeCN) so effective in a majority of cases. Use of a neutral buffer (a UV-transparent, volatile one is available)1 alleviates problems due to acidity but aggrevates those due to precipitation. And many proteins that do behave well on HPLC may balk when treated with a protease: they are either insoluble in suitable buffers or present a refractory surface with susceptible cleavage sites buried. The usual stratagem is to include a denaturant, but the level of urea that trypsin and most other enzymes will tolerate is often insufficient to promote complete and repoducible digestion, and detergents — which also are not always effective — interfer with separations employing RP-HPLC. An alternative to adjusting conditions to try to accomodate each ill-behaved protein is to modify these proteins at the start in such a way that they are all alike in their properties and common problems are overcome. Ideally this modification should be specific, complete, stable, universally produced under gentle conditions, unrestrictive with regard to other manipulations, and advantageous during sequencing.

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. Subtration of the estimated contribution of the oligosaccharide portion of the molecule leaves a M r 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 (11), which encodes a 35,500 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 (11).

Nearly all ascarosides in the ovaries ofA. lumbricoides occurred as esters. Among 15 identified esters, 13 occurred at concentrations exceeding 0.1% of the total ascarosides. Each ester consisted of one or two moles of glycone (ascarylose) and several homologs of long chain aglycone. The acid residues consisted of 95.6 mol % acetate and 4.4 mol % propionate. When present, propionate occurred only at the 2′ position of the glycone, which was always esterified. Esters with more than one free hydroxyl group were scarce, 89 wt % of the ascarosides being completely esterified. Monol aglycones contained 25–32 carbon atoms. The C29 homolog was most abundant, and ca. 25 wt% of these aglycones were branched and even-numbered. Diol aglycones were unbranched C29–35 homologs, C31 and C33 being about equally abundant in diol ascarosides and C33 most common in diol diascarosides. Within each ascaroside class the proportions of different homologs were correlated with the extent of esterification. Free ascarosides of the ascaroside layer of the egg shell, which are formed from the ovarian esters, consisted of 12% monol ascaroside, 10% diol ascaroside, 8% diol diascaroside and 70% diol ascaroside containing an acetylated aglycone. The absence from ascaroside esters of the α-methyl butyrate and α-methyl valerate residues which are plentiful in ovarian triglycerides and waxes suggests considerable biosynthetic specificity.

Anacystis nidulans flavodoxin is one of the long chain flavodoxins (Mayhew & Ludwig, 1975). Comparisons of its structure with the structures of shorter chain species (main text; Ludwig et al. , 1982) show that in A. nidulans flavodoxin most of the extra residues occupy a region adjoining the third helix and the fifth strand of parallel sheet. The sequences of peptides isolated after cyanogen bromide cleavage and after digestion with Staphylococcus aureus protease, reported here, fit into the electron density map of A. nidulans flavodoxin, starting near the middle of the third helix, and verify that the major insertion interrupts the fifth strand of parallel sheet.

A reliable procedure has been developed for the purification of the cytochrome c 1 aa 3 complex from the plasma membrane of T. thermophilus . The ratios heme C:heme A:Fe:C were found to be 1:2:3:2 confirming previous results, however, the molecular weight was found to be ∼92,000 rather than the ∼200,000 reported earlier [1]. Polyacrylamide gel electrophoresis under strongly denaturing conditions and high performance reverse phase liquid chromatography showed that cytochrome c 1 aa 3 is composed of only two subunits in 1:1 ratio. Both polypeptides have blocked N-termini. The smaller subunit (∼33,000) binds heme c and presumably no other metals. The larger subunit (∼55,000) is thus thought to contain the elements of cytochrome aa 3 and therefore be considered a single subunit cytochrome oxidase. The bacterial cytochrome c 1 aa 3 has been compared with beef heart cytochrome oxidase with a number of techniques including optical, EPR [1], Raman, MCD, and Mossbauer [2] spectroscopies. These experiments establish that the fundamental chemical properties of the redox centers are substantially similar in these two proteins. Cytochrome c 552 (from Thermus ), horse heart cytochrome c , and tetramethylphenylenediamine greatly stimulate the ascorbate oxidase activity of cytochrome c 1 aa 3 . This enhancement is characterized by a ‘high affinity’ component which results in only a small velocity increase and a ‘low affinity’ component which gives a large velocity increase. Very similar behavior has been previously observed with mammalian cytochrome oxidase [3]. Preliminary experiments show that vesicularized c 1 aa 3 is capable of proton pumping.