SEC11 is required for signal peptide processing and yeast cell growth

Among the collection of temperature-sensitive secretion mutants of Saccharomyces cerevisiae, sec11 mutant cells are uniquely defective in signal peptide processing of at least two different secretory proteins. At 37 degrees C, the restrictive growth temperature, sec11 cells accumulate core-glycosylated forms of invertase and acid phosphatase, each retaining an intact signal peptide. In contrast, other sec mutant strains in which transport of core-glycosylated molecules from the endoplasmic reticulum is blocked show no defect in signal peptide cleavage. A DNA fragment that complements the sec11-7 mutation has been cloned. Genetic analysis indicates that the complementing clone contains the authentic SEC11 gene, and that a null mutation at the SEC11 locus is lethal. The DNA sequence of SEC11 predicts a basic protein (estimated pI of 9.5) of 167 amino acids including an NH2-terminal hydrophobic region that may function as a signal and/or membrane anchor domain. One potential N-glycosylation site is found in the 18.8-kD (Sec 11p) predicted protein. The mass of the SEC11 protein is very close to that found for two of the subunits of the canine and hen oviduct signal peptidases. Furthermore, the chromatographic behavior of the hen oviduct enzyme indicates an overall basic charge comparable to the predicted pI of the Sec11p.     

Study of dominant symbiotic mutants of pea <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Phenogenetic studies of four symbiotic hypernodulating mutants of pea (Pisum sativum L.) induced from seeds of cultivar Rodno by chemical mutagen EMS were conducted. All mutants have improved symbiotic traits, i.e., an increased number of root nitrogen fixating nodules and high activity of nitrogenase. Symbiotic traits were shown to be inherited dominantly. Mutants grown in the field or in a greenhouse showed superiority over the original cultivar in productivity. An important feature of hypernodulating mutants was found that is responsible for the appearance of high-height productive plants in F₂ after crossing mutants and the original cultivar. Constant lines retaining the ability for high-level production up to the F₅ generation were created based on individual plants.     

Characterization of human apolipoprotein A-I expressed in Escherichia coli

Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)₆-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/l of culture medium. We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques. The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine. His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles. Lipid-bound native apoA-I and His-apoA-I showed very similar α-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I). The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes. In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values. Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I. The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture. In conclusion, we show that His-apoA-I expressed in E. coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux.     

Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes

Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.     

Identification of novel high molecular weight glutenin subunit mutants in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Bread wheat (Triticum aestivum L.) is a staple food crop eaten in different ways like pan and other food products. High molecular weight glutenin subunits (HMW-GS) are major determinants of the different wheat end-use qualities. Ethyl-methanesulfonate (EMS) mutagenized populations in plants can be used for the discovery of valuable mutants for basic research and breeding purposes. In this study, we report the identification of 27 HMW-GS M₃ mutants based on SDS-PAGE patterns from an EMS mutagenized population of the cultivar Baguette Premium 11. Nine mutations were detected in Ax2*, five in Bx7, four in By8, six in Dx5 and three in Dy10 subunit. Two Ax2* null mutants were characterized at molecular level finding in both cases premature stop codons associated. EMS would tend to generate more premature stop codons in glutenins genes than in others because these have a high frequency of glutamine codons. This type of mutation generates null alleles, therefore they are easily detectable by a low cost protocol like SDS-PAGE. The potential use of knock-out (null alleles) and SDS-PAGE size altered mutants for HMW-GS in wheat quality and nutrition is discussed.     

Physical Properties of Intact Proteins May Predict Allergenicity or Lack Thereof

Background

Predicting the allergenicity of proteins is challenging. We considered the possibility that the properties of the intact protein that may alter the likelihood of being taken up by antigen presenting cells, may be useful adjuncts in predicting allergens and non-allergens in silico. It has been shown that negatively charged acidic proteins are preferentially processed by dendritic cells.

Methodology

Datasets (aeroallergen, food-allergen and non-allergen) for in-silico study were obtained from public databases. Isoelectric point (pI), net charge, and electrostatic potential (EP) were calculated from the protein sequence (for pI and net charge) or predicted structure (for EP).

Result

Allergens and non allergens differed significantly in pI, net charge, and EP (p<0.0001). Cluster analysis based on these parameters resulted in well defined clusters. Non-allergens were characterized by neutral to basic pI (mean±SE, 7.6±0.16) and positive charge. In contrast allergens were acidic (5.7±0.15) and negatively charged. Surface electrostatic potentials calculated from predicted structures were mostly negative for allergens and mostly positive for non-allergens. The classification accuracy for non-allergens was superior to that for allergens. Thus neutral to basic pI, positive charge, and positive electrostatic potentials characterize non-allergens, and seem rare in allergens (p<0.0001). It may be possible to predict reduced likelihood of allergenicity in such proteins, but this needs to be prospectively validated.     

Mechanism of gene conversion in Ascobolus immersus

Summary Sixty two ascospore colour mutants have been induced in Ascobolus immersus: 25 by an acridine (ICR₁₇₀), 18 by N-methyl-N-nitro-N-nitrosoguanidine (NG) and 19 by ethyl methanesulfonate (EMS). All these mutants have been crossed to the wild type strain and their conversion spectrum has been determined. It appears that the conversion spectrum is closely related to the origin of the mutants studied with respect to the mutagen by which they were induced. All NG mutants gave numerous asci with postmeiotic segregation and an excess of conversion to wild type over conversion to the mutant type. ICR mutants gave no postmeiotic segregation and an excess of conversion to the mutant type. The majority of EMS mutants behave like NG mutants, but some showed only meiotic segregation with either an excess of conversion to the mutant type or an excess of conversion to wild type. These data are in good agreement with the hypothesis that the nature of the mutation has a strong influence on the conversion spectrum.     

Function of membrane proteins coupled to bacteriochlorophyll synthesis: Studies with wild type and mutant strains of Rhodopseudomonas spheroides

Membrane proteins with estimated molecular weights of 26,000, 22,000, 19,000, and 10,000–5,000 (designated 9, 10, 11, and 15, respectively) were found previously to be coupled to the synthesis of bacteriochlorophyll in Rhodopseudomonas spheroides. They have been attributed to the reaction center complex (proteins 9, 10, 11) and light-harvesting forms (protein 15) on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane fractions from the wild type and by analysis of certain photosynthetic mutants. Three of the mutants form light harvesting but not reaction-center bacteriochlorophyll; their membranes lack proteins 10 and 11, though proteins 9 and 15 are detectable. These mutants have little or no photophosphorylation or light-induced transhydrogenase activities and their respiration is not inhibited by illumination. Another mutant, strain 71-20 apparently has functional reaction centers, as determined by the above criteria, yet it does not grow anaerobically in the light for unknown reasons.     

Loop mutations can cause a substantial conformational change in the carboxy terminus of the ferritin protein.

Although some protein folding theories sustain that the peptides (loops) that connect elements of more compact secondary structure may be important in the folding process, most of the data accumulated until now seems to contradict this notion. To approach this problem we have isolated and characterized a number of mutants in which the amino acid sequence of the peptide that connects helix D and helix E in the H-chain of human ferritin has been randomized. Our results indicate that, though no single loop residue is absolutely required for ferritin to attain the native conformation, most of the mutants that we have obtained by random regional mutagenesis, affect its folding/assembly process. This conclusion was reached utilizing a sensitive test that associates the color formed by a colony synthesizing a hybrid ferritin-beta-galactosidase protein to the ability of the ferritin domain to fold and assemble as the native protein. The characterization of the folding/assembly properties of our collection of mutants and the comparison of the mutant loop sequences, have allowed us to draw the following conclusions. Mutants that have positively charged residues at position 159, 160 or 161 fail to assemble into the native protein shell and form an insoluble aggregate. Interestingly some loop amino acid sequences cause the E-helix to reverse direction and to expose its COOH group, normally hidden inside the protein cavity, to the solvent. The propensity of a given ferritin mutant to fold into this "non-native" conformation can be attenuated by the introduction of Gly at position 159 and 164, as in the natural ferritin.     

Isolation of plasmid DNA sequences that complement Rhodobacter sphaeroides mutants deficient in the capacity for CO2-dependent growth

Mutants deficient in the proper regulation and derepression of ribulose-1.5-bisphosphate carboxylase oxygenase (RuBPC/O) in Rhodobacter sphaeroides were isolated by ethyl methanesulphonate (EMS) and Tn5 mutagenesis of a recA parental strain. Mutants were identified by their ability to grow under conditions where the organism requires basal levels of RuBPC C/O for growth yet fail to grow under conditions which require derepression of the enzyme (Aut-). The newly isolated Aut- mutants exhibited phenotypes distinguishable from the previously isolated Aut- mutant, strain KW25/11. Rocket immunoelectrophoretic examination of RuBPC/O levels revealed marked variance in the ability of mutants to derepress form I and form II RuBPC/O in the absence of exogenous carbon. Evidence that some of the mutants possessed different mutations was substantiated by complementation of the EMS-generated mutants by entirely different genes isolated from a genomic library of R. sphaeroides constructed in the broad-host-range cosmid vector pVK102. Southern hybridization analysis of the complementing library isolates showed the complementing genes to be normally carried on the endogenous plasmids of R. sphaeroides. The gene complementing mutant strain KW25/11 was mapped by Tn5 insertional inactivation and the complementing region found to reside on a 1.5 kb PstJ. BamHI fragment. Complemented strains were unable to match wild-type levels of RuBPC/O under conditions requiring derepression of the enzyme, except for mutant strain EMS45. The Aut- phenotype, represented by the mutants isolated in this study, stems from a deficiency in some aspect of photoautotrophic growth.     

The Characterization of α-Glycerophosphate Dehydrogenase Mutants in DROSOPHILA MELANOGASTER

Thirty mutants of α-glycerophosphate dehydrogenase (αGPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of αGPDH mRNA in several of the mutants has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM^- by solution immunoprecipitation experiments, but of these, only three are CRM^- by a more sensitive ¹²⁵ I-protein A-based radioimmune gel assay. The viability of the three CRM^- mutants suggests that the absence of αGPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, αGpdh^nGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.     

Effect of proline residues on protein folding

Conformational energy calculations have been used to study the role of the proline residues in the folding of bovine pancreatic trypsin inhibitor. In the calculation, each of the four proline residues of this small protein is forced from the trans to cis peptide isomer while still part of the native folded structure. The cis proline residue can always be accommodated by small changes of the native conformation (< 1 Å root-mean-square deviation). For three of the four proline residues, Pro2, Pro9 and Pro 13, being in the cis form is calculated to destabilize the folded conformation by less than 11 kcal/mol, suggesting that rapid folding to a stable native-like conformation can occur with either isomeric form. For one of these three, Pro13, the destabilization is only 1 kcal/mol, suggesting the existence of an alternative folded native conformation with Pro13 cis. The fourth proline residue, Pro8, is calculated to destabilize the native conformation by so much (33 kcal/mol) that it will block folding in the manner proposed by Brandts et al. (1975).     

Multiple forms of phycoerythrin-545 from Cryptomonas maculata

Three multiple phycoerythrin-545 forms were purified from crude extracts of Cryptomonas maculata by preparative isoelectric focusing. The phycoerythrin forms are charge isomers with isoelectric points at 7.83, 5.05 and 4.84. The multiple pigment forms have similar molecular weights of 44500 daltons and are composed of subunits of unequal size in a 1:1 stoichiometry with molecular weights of () 9900 and () 15700 daltons, twice. The proposed quarternary structure of the native pigments is ()₂()₂.The charge differences of the phycoerthrins are caused by a charge heterogeneity of the light subunits, as revealed by urea gel electrophoresis. The chains of pigment form pI 7.83 had a greater electrophoretic mobility than those subunits of the acidic pigment forms pI 5.05 and pI 4.84.The phycoerythrin forms have an absorption spectrum with similar absorption maxima at 544 nm, but differ in the position of the long wavelength shoulders lying at 555 and 557 nm in the negatively charged pigment forms and at 560 nm for the phycoerythrin form with a pI at 7.83.The fluorescence emission spectra coincide in their asymmetrical shape with shoulders at about 620 nm; they slightly differ int he position of the emission maxima at 586 nm for the phycoerythrins with pIs at 4.84 and 5.05 and at 584 nm for phycoerythrin with pI at 7.83.Abbreviations PC phycocyanin - PE phycoerythrin - pI isoelectric point - SDS sodium dodecyl sulphate     

Functional role of the distal valine (E11) residue of alpha subunits in human haemoglobin

We have expressed human alpha-globin to a high level in Escherichia coli as a fusion protein, purified it and removed the N-terminal leader sequence by site-specific proteolysis with blood coagulation factor Xa. The apo globin has been refolded and reconstituted with haem and native beta-globin to form fully functional haemoglobin (Hb) with properties identical to those of native human Hb. By site-directed mutagenesis we have altered the distal residues of the alpha subunits and compared the functional properties of these mutant proteins. The rates of various ligands binding to these proteins in the R-state have been reported by Mathews et al. Here, we present the oxygen equilibrium curves of three E11 alpha mutants and the crystal structures of two of these mutants in the deoxy form. Replacing the distal valine residue of alpha-globin with alanine, leucine or isoleucine has no effect on the oxygen affinity of the protein in either quaternary state, in contrast to the equivalent mutations of beta subunits. The crystal structure of the valine E11 alpha----isoleucine mutant shows that the larger E11 residue excludes water from the haem pocket, but causes no significant movement of other amino acid residues. We conclude that the distal valine residue of alpha-globin does not control the oxygen affinity of the protein by sterically hindering ligand binding.     

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast)

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.     

Effect of enzymatic methylation of yeast iso-1-cytochrome c on its isoelectric point

Yeast iso-1- unmethylated and methylated apocytochrome c were synthesized in vitro by translating yeast cytochrome c mRNA, and by subsequently methylating the protein product. Unmethylated and methylated iso-1-holocytochrome c were extracted from Saccharomyces cerevisiae. By employing a column isoelectrofocusing technique, the pI values of these proteins were determined. The pI values of unmethylated and methylated apocytochrome c were found to be 9.60 and 8.70, respectively, with a difference of 0.90 pI unit. On the other hand, the pI values of unmethylated and methylated holocytochrome c were 9.72 and 9.68, respectively, with a difference of 0.04 unit. Therefore, although the pI values of both apo- and holocytochrome c decreased by methylation, methylation of apocytochrome c had a more profound effect on the pI of the protein. The result also indicated that conjugation of heme to apocytochrome c increased its pI value, resulting in the more "compact" and basic structure of the protein. The observed magnitude of the pI change subsequent to the methylation of apocytochrome c (decrease of 0.90 unit) seemed to be contradictory to the predicted increase in the value, since the positive charge is fixed on the quaternary amino group of trimethyllysine and there is no proton to titrate. Trimethylation of epsilon-NH2 group of Res-72 lysine of apocytochrome c could disrupt any possible hydrogen bond formed by the nitrogen atom of Res-72 lysine residues, as visualized by a space-filling model. The model and observed shift in the "effective charge" of the protein strongly suggest that conformational change in the apoprotein takes place upon methylation. This presumably altered conformation along with the decrease in pI caused by methylation may play a role in enhancement of apocytochrome c import into mitochondria.     

Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.     

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.     

Properties of isolated human alpha1-antitrypsins of Pi types M, S and Z.

1. alpha1-Antitrypsin contains a single thiol group partly blocked in native plasma and reactive after mild reduction. 2. Human alpha1-antitrypsins of Pi types F, M, S and Z have been isolated with native microheterogeneity using thiol-disulfide (SH-SS) interchange reactions utilizing the reactive thiol group. 3. The pI of the various microheterogeneous fractions are given for protein M. Stepwise desialylation of alpha1-antitrypsin indicates that the charge difference between the major fractions is one sialic acid residue between each. This is further supported by the pI changes obtained on substitution of the single thiol with positively or negatively charged compounds. 4. Desialyation of purified proteins from each Pi type converts the individual microheterogeneous fractions to one major fraction. The pI shift for the variants studied indicate a difference of plus or minus one or two charge units between protein M and the variants. 5. A difference of one sialic acid residue was obtained for proteins M and Z by the thiobarbituric assay, but stepwise removal of sialic acid with neuraminidase revealed almost identical stepwise change of pattern of both proteins indicating the same number of sialic acid residues. 6. Electrofocusing has been used to identify CNBr fragments from proteins M, S and Z. 7. An amino acid substitution has been found to be located in one of the eight CNBr fragments, glutamic acid in protein M is substituted by lysine in protein Z. 8. The average concentration of alpha1-antityprsin in plasma from healthy males was found to be 1.32 g/1.