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 alpha-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of gamma-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.     

Role of carbohydrate in biological functions of Friend murine leukemia virus gp71.

Purified gp71 of Friend murine leukemia virus (FLV) can interfere with virus infection, absorb neutralizing antibody, and in the presence of group-specific anti-gp71 antibody, hemagglutinate sheep erythrocytes. Interference by FLV gp71 with several murine leukemia viruses (MuLV) was tested in the XC and S + L- assay systems. Treatment of gp71 with trypsin or Pronase eliminated its interfering capacity. However, treatment with neuraminidase or a mixture of glycosidase enzymes, which left the major serological properties of gp71 intact, did not reduce the interference potential of gp71 for FLV or AKR MuLV. The capacity of gp71 to absorb type- or group-specific virus-neutralizing antibodies was similarly affected by the various enzyme treatments. In contrast, indirect hemagglutination by gp71 was abolished not only by proteases but also by treatment with glycosidase enzymes, although neuraminidase had no effect. Preliminary data indicate that infectivity of FLV or xenotropic MuLV was not affected by short treatment with glycosidase enzymes.     

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.     

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.     

Binding characteristics of Rauscher leukemia virus envelope glycoprotein gp71 to murine lymphoid cells.

The major envelope glycoprotein (gp71) purified from Rauscher leukemia virus (R-MuLV) binds efficiently to murine lymphoid cells but not to either murine nonlymphoid cells or lymphoid cells from other species. Binding of 125I-labeled R-MuLV gp71 was competitively inhibited by unlabeled glycoprotein, as well as by whole R-MuLV, but not by murine xenotropic viruses, R-MuLV p30, and several unrelated proteins. Polyacrylamide gel electrophoresis profiles of iodinated gp71 after binding to lymphoid cells were similar to prebound profiles. Antibody to R-MuLV gp71 prevented binding, whereas normal serum had no effect. Adsorption of the glycoprotein to murine lymphoid cells occurs rapidly and is time and temperature dependent. The procedure described is sensitive for detecting the binding activity of approximately 10(4) cells. Binding was proportional up to 2.5 X 10(5) cells per ml and plateaued above 10(7) cells per ml. In the presence of excess R-MuLV gp71, BALB/c thymocytes bound approximately 2.4 X 10(4) molecules per cell.     

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.     

A 2.4-kilobase-pair fragment of the Friend murine leukemia virus genome contains the sequences responsible for friend murine leukemia virus-induced erythroleukemia.

Friend murine leukemia virus (F-MuLV) is a replication-competent, ecotropic, NB-tropic retrovirus which produces a rapidly fatal erythroleukemia in susceptible strains of mice. We previously molecularly cloned the entire F-MuLV genome. Transfection of this cloned DNA into NIH 3T3 mouse fibroblasts produces a virus with the same leukemia-inducing characteristics as F-MuLV. To identify which portion of the F-MuLV genome is responsible for causing leukemia, we made recombinant viruses between subgenomic fragments of F-MuLV DNA and another retrovirus--Amphotroph clone 4070. Amphotroph clone 4070 is a replication-competent, amphotrophic, N-tropic virus which does not produce any detectable malignancy in mice. A 2.4-kilobase-pair fragment of F-MuLV DNA was isolated. This DNA fragment encompassed approximately 700 base pairs from the 3' end of the F-MuLV pol gene and 1.7 kilobase pairs of the env gene including all of gp70 and the N-terminal four-fifths of p15E. A molecularly cloned fragment of Amphotroph DNA was ligated to the 2.4-kilobase-pair F-MuLV DNA, and an 8.3-kilobase-pair hybrid F-MuLV-Amphotroph DNA was subcloned into a new plasmid (p5a25-H). Transfection of p5a25-H DNA into fibroblasts resulted in the production of a replication-competent, ecotropic, N-tropic retrovirus--5a25-H virus. Inoculation of this virus into newborn NIH Swiss mice caused leukemia within 4 to 6 months. The disease caused by 5a25-H was pathologically and histologically indistinguishable from the disease caused by F-MuLV. We conclude that the F-MuLV sequences needed to cause disease are contained in these 2.4 kilobase pairs of DNA.     

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 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.     

Inhibition of friend murine leukemia virus production by low-ionic-strength medium.

The effect of medium of low ionic strength on the release of virus from Friend leukemia cells has been studied. The release of infectious Friend leukemia virus is almost completely inhibited in medium of low ionic strength, as measured by a focus-forming assay (XC assay), by endogenous RNA-dependent DNA polymerase activity of released virus particles, and by electron microscope studies of the production of C-type particles. Friend leukemia virus-transformed proerythroblasts undergo extensive morphological changes in low-ionic-strength medium. The cells are viable in this medium, but they can no longer be stimulated with dimethyl sulfoxide to produce hemoglobin and increase virus production. Infectious virus is released between 30 and 120 min of resuspension of inhibited cells in normal medium. The rate of virus release after reversal of the inhibition is much greater than the rate of virus release during normal cell growth. The morphological changes occurring after dimethyl sulfoxide stimulation of Friend leukemia cells are compared with those resulting from resuspension in normal medium of cells inhibited by low ionic strength.     

The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence

The amino acid sequence of the large cyanogen bromide fragment (residues 11 to 127) derived from the NH2-terminal half of alpha-tropomyosin has been determined. This was achieved by automatic sequence analysis of the whole fragment as well as manual sequencing of fragments derived from tryptic digestion of the maleylated fragment and thermolytic, Myxobacter 495 alpha-lytic and Staphylococcus aureus protease digestion of the unmodified fragment. Methionine-containing overlap peptides have been isolated from tryptic digests of the maleylated protein as well as from S. aureus protease digests of the unmodified protein. Coupled with previously published information on the small cyanogen bromide fragments and methionine sequences of tropomyosin, these analyses have permitted the completion of the primary structure of the protein. The complete sequence differs by only 1 residue (Gln-24 instead of Glu-24) from that previously reported. Analysis of the sequence by several authors has permitted rational explanations for the stabilization of its coiled-coil structure, for the existence of its two chains in a nonstaggered arrangement, for a head-to-tail overlap of molecular ends of 8 to 9 residues, for the existence of 14 actin-binding sites on each tropomyosin molecule, and a suggestion for the site of binding of troponin-T.     

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

The receptor for human urokinase-type plasminogen activator (u-PA) was purified from phorbol 12-myristate 13-acetate-stimulated U937 cells by temperature-induced phase separation of detergent extracts, followed by affinity chromatography with immobilized diisopropyl fluorophosphate-treated u-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported. The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its amino-terminal fragment; 2) the identical electrophoretic mobilities observed for cross-linked conjugates, formed between either the purified protein or the u-PA receptor on intact U937 cells and the above ligands; 3) the identity of the apparent molecular weight of the purified protein to that predicted for the u-PA receptor in the same cross-linking studies; 4) the identical extent of glycosylation of the purified protein and of the u-PA receptor in crude membrane fractions, as detected after cross-linking; 5) the ability of antibodies raised against the purified protein to inhibit cellular binding of the amino-terminal fragment of u-PA.     

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.     

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.