Isolation and NH2-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors

Three N-glycosylated carrier proteins (CP) for insulin-like growth factors (apparent molecular weights 30-32, 42 and 45 kDa) were isolated from adult rat serum. They share the same amino terminus (up to amino acid 31) and are constituents of the growth hormone-dependent native 150-200 kDa IGF carrier complex. Residues 12-31 display 60 and 50% sequence homology, respectively, to residues 2-21 of fetal rat and to residues 4-22 of a human amniotic fluid IGF carrier protein. No homology exists with the type I or II IGF receptors. Adult rat serum also contains a fourth IGF CP (24 kDa) whose 9 NH2-terminal amino acids are identical to those of the fetal form. Our findings suggest that the three N-glycosylated components originate from the same IGF carrier protein (adult form) and that the 24 kDa protein is a separate (fetal) species.     

NH2-terminal amino acid distribution and amino acid composition of Streptococcus faecalis R soluble and ribosomal proteins

The NH(2)-terminal amino acid distribution of Streptococcus faecalis R soluble and ribosomal proteins isolated from cells at different stages of growth on either folate-sufficient or folate-deficient medium was determined by the dinitrophenyl method. The NH(2)-terminal residues do not follow the random distribution observed for the total amino acid composition of S. faecalis soluble and ribosomal proteins. Methionine and alanine occur most frequently; serine, threonine, aspartic and glutamic acids, and glycine are also present at the NH(2)-terminal position of S. faecalis R proteins. The absence of folic acid yields cells that are incapable of formylating methionyl-transfer ribonucelic acid tRNA(f) (Met), but does not affect either the qualitative or quantitative NH(2)-terminal distribution of total soluble or total ribosomal proteins compared to cells grown with folate. A small quantitative difference was observed in the frequency of distribution of certain amino acids at the NH(2)-termini between log and stationary phase soluble proteins. The amino acid residues found at the NH(2)-terminal position of S. faecalis proteins are qualitatively similar to those reported for several other organisms.     

Identification and NH2-terminal amino acid sequence of three insulin-like growth factor-binding proteins in porcine serum.

Three distinct species of IGFBP in porcine serum were identified by NH2-terminal amino acid sequence analysis. The IGFBPs identified include pIGFBP-2 (34 kDa), three isoforms of pIGFBP-3 (43, 40 and 30 kDa) and two isoforms of pIGFBP-4 (30 and 26 kDa). The three isoforms of pIGFBP-3 were found to have a common NH2-terminal amino acid sequence, as were the two isoforms of pIGFBP-4. These results indicate that porcine serum contains a truncated form of IGFBP-3 and two forms of pIGFBP-4, similar to those previously isolated from human and rat serum. Furthermore, the presence of a truncated form(s) of the GH-dependent IGFBP-3 in porcine serum suggests that elucidating its origin and function may be important in understanding how IGFBPs affect the somatogenic actions of GH.     

Amino acid composition and NH2-terminal amino acid sequence of rat platelet secretory phospholipase A2

The amino acid composition and partial NH2-terminal amino acid sequence of the phospholipase A2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH2-terminal 24 amino acids were found to be as follows: X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp- Val-Ser-Tyr-Gly-Phe-Tyr-Gly- The enzymes contains 5Phe, 8Met, 9Ile, 24Tyr, and 25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A2. This is the first report of the tentative characterization of a eukaryotic phospholipase A2, the cellular source of which is known, i.e., it does not originate from a venom or the pancreas.     

Structural proteins of Western equine encephalitis virus: amino acid compositions and N-terminal sequences

The structural proteins of Western equine encephalitis virus, a member of the alphavirus group, have been characterized by the determination of their amino acid compositions and by N-terminal sequence analysis. More than 60 residues of the N-terminal sequences of each of the envelope glycoproteins have been determined. A comparison of these sequences with the previously determined sequences of two related alphaviruses. Sindbis virus and Semliki Forest virus, strongly supports the view that all three viruses have evolved from a common ancestor and provides information on the pattern of this evolution. The analysis of the capsid proteins of Western equine encephalitis virus shows that the nucleocapsid of this virus can accommodate a considerable degree of variability in its protein component and that at least some regions of alphavirus capsid proteins show more extensive differences between different viruses than do the envelope glycoproteins.     

Glycoproteins of friend murine leukemia virus: separation and NH2-terminal amino acid sequences of gp69 and gp71.

The NH2-terminal amino acid sequences (initial 23 residues) of Friend murine leukemia virus gp71 and gp69 were determined and found to be different but highly related. Friend murine leukemia virus gp71 differed from Rauscher murine leukemia virus gp70 in only one position. Friend murine leukemia virus gp69 showed approximately 41% homology to these glycoproteins but lacked the glycosylation site (sequon) occurring at position 12 in Rauscher murine leukemia virus gp70.     

NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii

A precursor (pS) to the small subunit (S) of ribulose1-,5-bisphosphate carboxylase is the major product of cell-free protein synthesis directed by poly(A) containing RNA from Chlamydomonas reinhardtii. We present sequence data for in vitro-synthesized pS, for in vitro- synthesized S that in generated from pS by posttranslational incubation with a Chlamydomonas cell extract, and for in vitro-synthesized, mature S. We show that pS contains an NH2-terminal extension of 44 amino acid residues that is removed by cleavage at the correct site when pS is converted to S by an endoprotease present in the Chlamydomonas cell extract.     

NH2-terminal analysis of Wolfgram and Folch-Lees proteolipid proteins

Abstract— The NH₂-terminal amino acids of Wolfgram and Folch-Lees proteolipids of bovine and human CNS myelin were determined using the cyanate method (Starke & Smyth , 1963) followed by direct amino acid analysis of the products. Glycine predominated in every case and was recovered in amounts similar to the results described by Whikehart & Lees (1973), who used a dansylation technique followed by thin layer chromatography of the DNS-amino acids. In the present study substantial amounts of glutamic acid, serine, alanine and aspartic acid were also recovered, plus traces of other amino acids. Few differences were observed between Wolfgram and Folch-Lees proteolipids. The end group products of purified W1 proteolipid of bovine Wolfgram fraction, of diazometholysed Folch-Lees proteolipid, and of a sample of phosphatidyl serine had essentially the same composition. The similarity of these results, especially for both fractionated and unfractionated Wolfgram proteolipid, may be evidence that the observed products are derived from phosphoglycerides present in proteolipid rather than from the actual NH₂-terminals of the protein.     

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.     

Antibodies to an NH2-terminal myristoyl glycine moiety can detect NH2-terminal myristoylated proteins in the retrovirus-infected cells

Novel antibodies were raised against a synthetic NH2-terminal myristoyl glycine moiety which is characteristic of N-myristoyl-proteins. Antisera raised against N-myristoyl-Gly-hemocyanin reacted with N-myristoyl-Gly-[125I]albumin. The immunoreaction was competed for by albumin conjugated with N-myristoyl-glycine, while underivatized albumin had no effect. Of the [3H]myristate-labeled proteins detected, pp60v-src, which is a transforming protein of Rous sarcoma virus, and p19gag and p17gag, which are core proteins in the human T-cell leukemia virus and the human immunodeficiency virus, were identified as N-myristoylated proteins by the radioimmunoprecipitation analyses with the antibody.     

The NH2-terminal amino acid sequence of human proparathyroid hormone by radioisotope microanalysis.

The amino terminal amino acid sequence of human proparathyroid hormone was determined by a highly sensitive radioisotope method. Fresh human parathyroid glands were incubated with one of several ³H-labeled amino acids after which human proparathyroid hormone labeled with [³H]lysine, [³H]serine, [³H]valine, [³H]arginine, or [³H]leucine was isolated. These specimens were subjected to several cycles of Edman degradation. An increase in the amount of radioactivity liberated at any cycle was taken as evidence that the amino acid at that cycle was the one with which the sample was labeled. By this approach, we found that the amino-terminal sequence of human proparathyroid hormone is lys¹-ser²-val³-lys⁴-lys⁵-arg⁶-ser⁷-val⁸-ser⁹ … leu¹³ … leu¹⁷ … lys¹⁹ … Based on these results, we conclude that the amino-terminal region of human proparathyroid hormone consists of a hexapeptide lys-ser-val-lys-lys-arg followed by the amino acid sequence of human parathyroid hormone.     

Amino acid composition and NH2-terminal amino acid sequence of human phospholipase A2 purified from rheumatoid synovial fluid

The amino acid composition and partial NH2-terminal amino acid sequence of an extracellular phospholipase A2 in human rheumatoid synovial fluid were determined. The predominant amino acids in the phospholipase A2 were cysteine, glycine, arginine, and lysine, suggesting that it is a basic one. The NH2-terminal 34 amino acids were found to be as follows: Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu- Ser-Tyr-Gly-Phe-Tyr-Gly-Cys-X-Cys-Gly-Val-Gly-Gly-Arg-Gly The enzyme contains Phe-5, Met-8, Ile-9, Tyr-24, Gly-25, Cys-26, Cys-28, Gly-29, Gly-31, Gly-32, and Gly-34 residues, all of which are conserved in most of the sequenced phospholipase A2. The remarkable feature of this enzyme was the absence of Cys-11, which is conserved in the "Group I" enzyme family. This is the first report concerning partial amino acid sequences of human non-pancreatic phospholipase A2.     

A comparative study on the NH2-terminal amino acid sequences and some other properties of six isozymic forms of human pepsinogens and pepsins

Six pepsinogen isozymogens, including five forms of pepsinogen A (PGA) and an apparently single form of pepsinogen C (PGC), were isolated simultaneously from the purified total pepsinogen fraction of human gastric mucosa by fast protein liquid chromatography on a Mono Q column, and their NH2-terminal amino acid sequences and some other properties were compared. Upon activation at pH 2.0, all the isozymogens were converted to the corresponding pepsins in a stepwise manner through intermediate forms. The activation rates and the cleavage sites in the activation peptide segment to generate intermediate forms were significantly different among the isozymogens. The NH2-terminal 85-residue amino acid sequences of these isozymogens were determined, including the sequences of the activation peptide segments and the NH2-terminal regions of the corresponding pepsins. Differences in amino acid sequence were found at positions 43 and 77 among the pepsinogen A isozymogens; the residue at position 43 was Lys in PGA-5, PGA-4, and PGA-3a, and Glu in PGA-3 and PGA-2, and the residue at position 77 was Leu in PGA-5 and PGA-4 and Val in PGA-3 and PGA-2. Phosphate was not found in any of the isozymogens. The corresponding pepsins also showed significant variations in properties such as specific activities toward synthetic and protein substrates, pH dependence of activity, susceptibility to various inhibitors, and thermal and alkaline stabilities.     

Polypeptide Compositions and NH2-terminal Amino Acid Sequences of Proteins in Foxtail and Proso Millets

Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH₂-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1% and 80.0% prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27-19 kDa were rich in leucine and alanine, whereas the 17-14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH₂-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to α-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of γ-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.