Evidence for a change in catalytic properties of glyceraldehyde 3-phosphate dehydrogenase monomers upon their association in a tetramer

The complete amino acid sequence of human spleen apoferritin has been determined. It consists of 174 amino acids, corresponding to M_r20017. The sequence is very similar to that of horse spleen apoferritin (14% difference between the two sequences). Some peptides were isolated and sequenced which could not be placed in the sequence but which are homologous with part of the principal sequence. Automatic sequence determination of a large peptide resulting from acid cleavage allows us to establish the presence of two homologous sequences (in the ratio $\text{80}\text{20}$).     

Aspartate transaminase from E. coli: amino acid sequences of the NH2-terminal 33 residues and chymotryptic pyridoxyl tetrapeptide.

The amino acid sequences of pyridoxal-binding tetrapeptide and the NH₂-terminal portion of aspartate transaminase from $\text{E.}$$\text{coli}$ B were analyzed and compared with those of the corresponding parts of the cytosolic and mitochondrial isozymes from pig heart. After borohydride reduction and chymotryptic digestion of the $\text{E.}$$\text{coli}$ enzyme, a pyridoxal-containing peptide was isolated, showing the sequence, Ser-Lys(Pxy)-Asn-Phe, identical with that of the cytosolic isozyme. The NH₂-terminal sequence was determined up to 33 residues with a liquid phase sequence analyzer. Nearly the same degree of homology was observed among the NH₂-terminal sequences of the three aspartate transaminases.     

Sequential homology of the collagenase from eucaryote Hypoderma lineatum with the proteinases of the trypsin family

The sequence of 32 N-terminal amino acid residues of the collagenase from the larvae Hypoderma lineatum of mol. weight 24000 is homologous with the sequences of pancreatic proteinases. This analysis confirms the hypothesis of similarity made on the basis of amino acid composition, physico-chemical properties and inhibition studies. It is proposed that within the trypsin-like family exists a group of eucaryote collagenases with digestive rather than morphogenic function which cleave specifically native collagen at $\text{3}\text{4}$ from its N-terminus.     

N-terminal amino acid sequences of acid proteases: acid proteases from Penicillium roqueforti and Rhizopus chinensis and alignment with penicillopepsin and mammalian proteases.

The amino-terminal sequence (33 residues) of the acid protease from Penicillium roqueforti has been determined with an automated sequencer. The amino-terminal sequence of Rhizopus pepsin (published by Sepulveda, P., Jackson, K. W. & Tang, J. (1975) Biochem. Biophys. Res. Commun. 63, 1106-1112) has been extended from 27 residues to 39 residues. Also, it was found that two forms of Rhizopus pepsin differ in position 15, where Rhizopus pepsin I has an isoleucine and Rhizopus pepsin II a valine residue. The new sequences have been aligned with the amino-terminal sequences of penicillopepsin (EC 3.4.23.7), pig pepsin (EC 3.4.23.1), calf chymosin (EC 3.4.23.4), human pepsin (EC 3.4.23.2), human gastricsin (EC 3.4.23.3), and cow pepsin (EC 3.4.23.1). Residues 31-35 (numbering based on pig pepsin, Tang, J., Sepulveda, P., Marciniszyn, Jr., J., Chen, K.S.C., Huang, W.-Y. , Tao, N., Liu, D. & Lanier, P. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3437-3739) are identical in all enzymes. This section contains one of the two aspartic acids (Asp-32) implicated in the active site. The similarity of the sequences provides strong evidence for the homology of these acid proteases.     

Structure of human luteinizing hormone beta subunit: evidence for a related carboxyl-terminal sequence among certain peptide hormones

Amino acid sequence analysis of human luteinizing hormone (lutropin) beta subunit has been carried out in order to reconcile several discrepancies apparent in previously published structures. Our sequence coincides closely with that proposed originally by Shome and Parlow (J.Clin.Endocr.Metab. $\text{36}$, 618–621, 1973). However, we found a unique solution to the carboxyl-terminal region (-Thr-Cys-Asp-His-Pro-Gln-OH) which is closely homologous with the corresponding segment of hCG and the animal LH beta subunits. The C-terminal three residues appear in closely similar sequences at the C-termini of several other peptide hormones. This suggests a primitive genomic relationship with conservation of this region during evolution of these otherwise divergent polypeptides.     

Proline inhibition of pyrroline-5-carboxylate reductase: differences in enzymes obtained from animal and tissue culture sources

The complete amino acid sequence for the 148 amino acid flavodoxin from $\text{Desulfovibrio}\text{vulgaris}$ is presented. This is the first flavoenzyme for which both the complete amino acid sequence and a 2.5 Å resolution x-ray diffraction structure are now known. The position of some important residues in the binding of FMN are given. The D. vulgaris sequence is compared with other published flavodoxin sequences.     

The amino acid sequence of the Azotobacter vinelandii flavodoxin.

The amino acid sequence of a group II flavodoxin, the $\text{Azotobacter vinelandii}$ flavodoxin has been determined. The FMN-redox protein was shown to exist as a single polypeptide chain and to contain 179 amino acids. Despite the rather low amino acid sequence homology with the other flavodoxins sequenced, it is concluded that sequences of the group I and group II flavodoxins are homologous. The major differences between the group I and group II flavodoxins appears to be a lengthening in the C-terminal region in the group II flavodoxins.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Partial amino acid sequence of mouse beta2-microglobulin.

A highly purified preparation of mouse β₂-microglobulin has been obtained from sodium thiocyanate extracts of liver cell membranes of $\text{A}\text{J}$ strain mice and the amino acid sequence has been partially determined. The first 40 residues have been assigned except for position 34. In the sequence, the mouse protein differs only at 9 positions from human β₂-microglobulin at 8 positions from the dog homologue and at 11 positions from the rabbit homologue. The sequence has also a homology to the constant regions of mouse γG_2a, most closely to the C_H3 region. These data support the conclusion that the mouse protein is indeed the mouse homologue of human β₂-microglobulin.     

Purification and characterization of cytochrome C3 (Mr 26,000) isolated from Desulfovibriodesulfuricans Norway strain

An acidic cytochrome c (P_i = 4.8) has been purified from $\text{Desulfovibrio}\text{desulfuricans}$ Norway. Its molecular weight was estimated to be 26,000 but a monomeric form of 13,500 molecular weight has been obtained. The comparison of its amino acid composition and N terminal sequence has characterized this cytochrome as a new cytochrome, different from cytochrome c₃ (Mr 13,000) and cytochrome c_553(550) studied in the same organism. Its optical spectrum was similar to cytochrome c₃ (Mr 13,000) accordingly it has 4 haems per subunit. The absence of absorption at 695 nm indicates that two histidine residues are implicated as fifth and sixth ligand for haem iron. This new cytochrome is homologous to the cytochrome C₃ (Mr 26,000) previously described for $\text{Desulfovibrio}\text{gigas}$ and $\text{Desulfovibrio}\text{vulgaris}$.     

DNA replication in a polymerase I deficient mutant and the identification of DNA polymerases II and 3 in Bacillus subtilis

The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation have been determined. These data indicate that the products of the first step of activation, whether derived from the action of factor Xa or thrombin, are identical. The data also show that the activation of prothrombin proceeds by the sequential cleavage of the amino terminal region of prothrombin and the intermediates, and confirm the mechanism of prothrombin activation as: NH₂-Prothrombin-COOH $\text{Xa or thrombin}$ NH₂-Intermediate 3 + Intermediate 1-COOH; NH₂-Intermediate 1-COOH $\text{Xa}$ NH₂-Intermediate 4 + Intermediate 2-COOH; NH₂-Intermediate 2-COOH $\text{Xa}$ NH₂-A chain α-thrombin -S-S-B chain α-thrombin-COOH.Previous reports from this laboratory have demonstrated that the activation of prothrombin proceeds through several single-chain intermediates prior to the appearance of thrombin activity. (1) Subsequent studies have sequence of the prothrombin molecule can be deduced from the sequences of its activation intermediates and we are continuing our studies toward this goal.     

Analysis of the primary structure of collagen for the origins of molecular packing

The amino acid sequence in the triplet region of the α1 chain of collagen was analyzed for complementary relationships that would explain the stagger of multiples of 670 Å between the rod-like molecules in the fibril. The analysis was done by moving the sequence of 1011 amino acids past itself and scoring for complementarity between opposing amino acids allowing a range of ±2 to 3 residues. It was found that interactions between amino acids of opposite charge and between large hydrophobic amino acids in the overlapping region between two chains are maximal when the chains are staggered by 0D, 1D, 2D, 3D and 4D, where D = 234 ± 1 residues. The residue repeat derived from this value is 2.86 ± 0.02 Å. The existence of a D separation between interacting residues was shown to be reflected in the actual distribution of large hydrophobic amino acids. Surprisingly, the distribution approximates the pattern ($\text{2D}\text{11}$)₅($\text{D}\text{11}$) repeated over 4.4D intervals. The regularity may arise from structural constraints imposed by super-coiling. The distribution of charged residues is less regular and does not show a well-defined periodicity. However, positively-charged residues tend to be near negatively-charged residues, allowing intramolecular charge neutralization as well as strong intermolecular charge interactions at 0D.     

Chou-Fasman analysis of the secondary structure of F and Le interferons

The Chou-Fasman method for calculating the secondary structure of $\text{F}$ and $\text{Le}$ interferons from their amino acid sequences predicts several regions of homologous structure in the two interferons. Those areas tend to be located in the middle and C- terminal ends of the molecules. Total predicted content of α helix is 55% for $\text{Le}$ interferon and 36% for $\text{F}$ interferon. Predicted β-pleated sheet residues total 33% for $\text{F}$ and 16% for $\text{Le}$.     

Structural studies of monoclonal human cryoprecipitable immunoglobulins

The light chain type, immunoglobulin class and when possible, heavy chain subclass of eleven monoclonal human cryoglobulins were correlated with the variable region subgroup of their light chains. The variable region subgroups were assigned by determining the primary amino acid sequence for the first 15 amino-terminal residues of these light chains. $\text{5}\text{5}$ IgM cryoglobulins which react with human IgG had light chains of the variable region-III kappa chain subgroup (vK-III). $\text{4}\text{4}$ IgG and $\text{2}\text{2}$ IgM cryoglobulins with undefined antibody specificity had both lambda and kappa light chains none of which were vK-III. The data support the concept that there is marked restriction of the IgM anti-IgG antibody response to the IgG auto-antigen.     

The amino acid sequence of carbonic anhydrase I from the rhesus macaque

The complete amino acid sequence of carbonic anhydrase I (CA I) isolated from the red cells of the rhesus macaque ($\text{Macaca}$$\text{mulatta}$) is presented. This sequence was obtained by aligning peptides derived from various fragmentation procedures with the fully characterized sequence of human CA I. When the peptides of rhesus CA I were ordered in this manner, 13 of the 260 residues were found to differ from the human CA I sequence. The known markedly higher specific esterase activity of rhesus CA I compared to human CA I could not be correlated with any changes in residues postulated to be within 10 Å of the single zinc ion at the active site.     

Structural studies on epsilon-prototoxin of Clostridium perfringens type D. Localization of the site of tryptic scission necessary for activation to epsilon-toxin.

The mechanism of activation of ε-prototoxin to ε-toxin has been ascertained from partial amino acid sequences of both ε-prototoxin and ε-toxin. The activation of ε-prototoxin from $\text{Clostridium perfringens}$ type D by brief exposure to trypsin is caused by scission of a peptide bond between Lys₁₄ - Ala₁₅. A small peptide (14 amino acid residues) is split from the NH₂-terminus of the ε-prototoxin to give the active ε-toxin.     

Oligosaccharide units of lysosomal cathepsin D from porcine spleen. Amino acid sequence and carbohydrate structure of the glycopeptides

The amino acid sequences near the glycosylation sites and the oligosaccharide structures have been determined for the lysosomal protease cathepsin D from porcine spleen. Cathepsin D light and heavy chains were separately digested with proteases and the glycopeptides were purified. A single sequence was constructed from the amino acid sequence of the light chain glycopeptides which is: Tyr-Asn-Ser-Gly-Lys-Ser-Ser-Thr-Tyr-Val-Lys-Asn(CH2O)-Gly-Thr-Thr-Phe. A single glycopeptide sequence was also obtained for the heavy chain: Lys-Gly-Ser-Leu-Asp-Tyr-His-Asn(CH2O)-Val-Thr-Arg-Lys-Ala-Tyr. The light chain sequence is homologous with the sequence of porcine pepsin from residues 56 to 71. The heavy chain sequence is homologous with the pepsin sequence from residues 176 to 189. Thus, the 2 oligosaccharide-linked asparagines in cathepsin D correspond to residues 67 and 183 in pepsin and other homologous aspartyl proteases. These positions are located on the surface of the crystal structures of aspartyl proteases. Five oligosaccharides linked to Asn-67 were separated and their structures determined with proton NMR. Four major oligosaccharides are structural variants from the high mannose-type having 3, 5, 6, and 7 mannoses, respectively. A minor structure contained a third GlcNAc. Three oligosaccharide structures were found linked to Asn-183. Two major oligosaccharides are of the high mannose-type each with 5 mannose residues. One of the two contains a fucose linked to a GlcNAc. A third, very minor oligosaccharide contains galactose.     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

The biosynthesis of phosphorylated tyrosine hydroxylase by organ cultures of rat adrenal medulla and superior cervical ganglia: a correction.

Determination of the complete amino acid sequence of the rubredoxin isolated from the sulfate reducing bacterium $\text{Desulfovibrio}$$\text{gigas}$ showed that the molecule consists of a single polypeptide chain of 52 residues. The sequence of the first 42 residues was determined using an automatic Protein Sequencer. Peptides derived from tryptic hydrolysis and from specific cleavage at tryptophan residue were used to construct the total sequence. Compared with the sequence of $\text{Desulfovibrio}$$\text{vulgaris}$ rubredoxin, 37 positions are identical, and with the sequences of $\text{Clostridium}$$\text{pasteurianum}$, $\text{Peptostreptococcus}$$\text{elsdenii}$, $\text{Micrococcus}$$\text{aerogenes}$ and $\text{D.}$$\text{vulgaris}$ rubredoxins, 20 matching residues occur. A crystallographic study of the $\text{D.}$$\text{gigas}$ rubredoxin is in progress.     

Inhibition of Human Pepsin and Gastricsin by α2-Macroglobulin

The inhibitory effects of human α₂-macroglobulin (α₂-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α₂-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α₂-M mainly at the His⁶⁹⁴-Ala⁶⁹⁵ bond and Leu⁶⁹⁷-Val⁶⁹⁸ bond, respectively, in the bait regions sequence of α₂-M. The conformation of α₂-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α₂-M.