Iron-sulfur cluster assembly: NifU-directed activation of the nitrogenase Fe protein

The NifU protein is a homodimer that is proposed to provide a molecular scaffold for the assembly of [Fe-S] clusters uniquely destined for the maturation of the nitrogenase catalytic components. There are three domains contained within NifU, with the N-terminal domain exhibiting a high degree of primary sequence similarity to a related family of [Fe-S] cluster biosynthetic scaffolds designated IscU. The C-terminal domain of NifU exhibits sequence similarity to a second family of proposed [Fe-S] cluster biosynthetic scaffolds designated Nfu. Genetic experiments described here involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation, although the N-terminal domain appears to have the dominant function. These in vivo experiments were supported by in vitro [Fe-S] cluster assembly and transfer experiments involving the activation of an apo-form of the nitrogenase Fe protein.     

Partial amino acid sequences of peptidyl-prolyl isomerases ofFusarium sporotrichioides

Peptidyl-proprylyl cis-trans isomerase (PPIase) activity was observed from crude extract ofFusarium sporotrichioides. Proteins from this fungi were separated by two-dimensional polyacrylamide gel electrophoresis and more than one thousand protein spots were separated. Two cytosolic PPIases were found by the N-terminal sequencing from the two separated spots. The N-terminal 41 residues of the major protein spot showed high sequence identity (63.4%) with PPIase fromNeurospora crassa. This protein was designated as PPIase a, having an apparent molecular mass of 20 kD and pI 7.0. The minor other protein spot, having a similar molecular mass but distinguishable pI 6.4, was also sequenced and the N-terminal twenty residues were almost identical to PPIase a and was designated as PPIase b.     

Flammin and velin: new ribosome inactivating polypeptides from the mushroom Flammulina velutipes

A protein designated flammin and exhibiting a molecular mass of 30kDa, and another protein designated velin and possessing a molecular mass of 19 kDa, were isolated from the fruiting bodies of the edible mushroom Flammulina velutipes. Flammin and velin inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 1.4 and 2.5 nM, respectively. Flammin demonstrated only a small degree of resemblance in N-terminal sequence to angiosperm type 1 ribosome inactivating proteins (RIPs) such as trichosanthin, alpha-momorcharin and beta-momorcharin but no sequence similarity to other mushroom RIPs. Velin manifested limited sequence homology to the A chain of abrin, a type 2 angiosperm RIP. Neither flammin nor velin showed any ribonuclease or protease activity. Both flammin and velin were unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-Sepharose. They were separable in gel filtration on Superdex 75 by fast protein liquid chromatography.     

Mungin, a novel cyclophilin-like antifungal protein from the mung bean

A protein designated mungin, isolated from mung bean (Phaseolus mungo) seeds, possessed activity against the fungi Rhizoctonia solani, Coprinus comatus, Mycosphaerella arachidicola, Botrytis cinerea, and Fusarium oxysporum. The 18-kDa protein also possessed a novel N-terminal sequence with similarity to cyclophilins. It exerts an inhibitory action against alpha- and beta-glucosidases suppresses [(3)H]thymidine in corporation by mouse splenocytes.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

IN VITRO ANTIVIRAL ACTIVITY OF AN ALKALINE TRYPSIN FROM THE DIGESTIVE JUICE OF Bombyx mori LARVAE AGAINST NUCLEOPOLYHEDROVIRUS

Alkaline trypsin protein of molecular mass 25,436 Da purified from the digestive juice of Bombyx mori larvae indicated strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) under in vitro conditions. Partial N-terminal amino acid sequence of the protein was determined and the cDNA was cloned based on the amino acid sequence. A homology search of the deduced amino acid sequence of the cDNA showed 55% identity with Helicoverpa armigera trypsin and the active site of this protein was completely conserved. Hence, the protein was designated B. mori trypsin (Bmtryp). The results suggest that Bmtryp, an insect digestive enzyme, can be a potential antiviral factor against BmNPV at the initial site of viral infection.     

Hispin,a novel ribosome inactivating protein with antifungal activity from hairy melon seeds

A ribosome inactivating protein demonstrating a molecular mass of 21 kDa and a novel N-terminal sequence was isolated from seeds of the hairy melon. The purification procedure involved affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose and Mono S. The protein designated hispin inhibited translation in the cell-free rabbit reticulocyte lysate system with an IC50 of 165 pM and exhibited N-glycosidase activity. Antifungal activity was also observed.     

Characterization of the small hydrophobic proteins associated with pulmonary surfactant

Lipid extracts of bovine pulmonary surfactant, which retain many of the biophysical characteristics of natural surfactant, contain approx. 98% lipid and 2% protein, as determined by amino acid analysis. Polyacrylamide/urea gel electrophoresis reveals that lipid extract surfactant contained a major apoprotein band with apparent Mr 3500 and minor apoprotein bands with apparent Mr 15,000 and 7000. After reduction, the 15 kDa band disappears and is replaced by a prominent band with apparent Mr = 5000. Reduction also results in a relative diminution of the 7 kDa band and a relative increase in the intensity of the 3.5-kDa band. Edman degradation reveals two major peptide sequences which have been designated surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptide (N-terminal Leu) and a minor sequence designated surfactant-associated peptide (N-terminal Ile). The latter surfactant-associated peptide appears to be related to the N-terminal Leu peptide but lacks the terminal Leu. N-Terminal analysis by dansylation demonstrates that the 15 and 5 kDa (reduced) apoprotein species contain N-terminal Phe, Leu and Ile. The 3.5 and 7 kDa bands contain only N-terminal Leu and Ile. Chromatography of lipid extracts on silicic acid columns gives rise to fraction I, which contains protein and phosphatidylglycerol, and fraction II, which contains protein, phosphatidylglycerol and phosphatidylethanolamine. Fraction I was primarily composed of the 15-kDa apoproteins, while fraction II contained mainly the 3.5 and 7 kDa apoproteins. Both fractions exhibited biophysical activity after reconstitution with dipalmitoylphosphatidylcholine. These results indicate that lipid extracts contain an oligomer of 15 kDa containing surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptides (N-terminal Leu or Ile) which interact through sulfhydryl and perhaps other bonds. Lipid extracts also contain 3.5 kDa monomers of surfactant-associated peptides with N-terminal Leu and N-terminal Ile which can dimerize through sulfhydryl and perhaps hydrophobic interactions.     

The S-layer protein from Campylobacter rectus: sequence determination and function of the recombinant protein

The gene encoding the crystalline surface layer (S-layer) protein from Campylobacter rectus , designated slp , was sequenced and the recombinant gene product was expressed in Escherichia coli . The gene consisted of 4086 nucleotides encoding a protein with 1361 amino acids. The N-terminal amino acid sequence revealed that Slp did not contain a signal sequence, but that the initial methionine residue was processed. The deduced amino acid sequence displayed some common characteristic features of S-layer proteins previously reported. A homology search showed a high similarity to the Campylobacter fetus S-layer proteins, especially in their N-terminus. The C-terminal third of Slp exhibited homology with the RTX toxins from Gram-negative bacteria via the region including the glycine-rich repeats. The Slp protein had the same N-terminal sequence as a 104-kDa cytotoxin isolated from the culture supernatants of C. rectus . However, neither native nor recombinant Slp showed cytotoxicity against HL-60 cells or human peripheral white blood cells. These data support the idea that the N-terminus acts as an anchor to the cell surface components and that the C-terminus is involved in the assembly and/or transport of the protein.     

Flammulin: a novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes.

A protein with a molecular weight of 40 kDa, capable of inhibiting cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 0.25 nM, was isolated from fruiting bodies of the mushroom Flammulina velutipes. The protein, designated flammulin, was devoid of ribonuclease activity. Flammulin was unadsorbed on DEAE-cellulose at neutral pH and low ionic strength and adsorbed on CM-Sepharose and Affi-gel blue gel under similar conditions. Its N-terminal sequence demonstrates sites of similarity to those of plant ribosome-inactivating proteins (RIPs).     

Isolation of pisumin,a novel antifungal protein from legumes of the sugar snap pea Pisum sativum var macrocarpon

An antifungal protein with a novel N-terminal sequence GVGAAYGCFG and a molecular mass of 31 kDa was isolated from the legumes of the sugar snap pea Pisum sativum var. macrocarpon. The protein, designated pisumin, exhibited antifungal activity against Coprinus comatus and Pleurotus ostreatus and much weaker activity against Fusarium oxysporum and Rhizoctonia solani. Pisumin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC(50) of 6 microM. Pisumin was similar to other leguminous antifungal proteins in that it was adsorbed on Affi-gel blue gel and CM-Sepharose.     

Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.     

FEM1, a Fusarium oxysporum glycoprotein that is covalently linked to the cell wall matrix and is conserved in filamentous fungi

As part of an investigation of the cell wall structure of plant pathogenic, filamentous fungi, we set out to characterize covalently bound cell wall glycoproteins (CWPs) of the tomato pathogen Fusarium oxysporum. N-terminal sequencing of an abundant 60-kDa CWP led to the cloning of the corresponding gene, which we have designated FEM1 (Fusarium extracellular matrix protein). The gene contains an ORF encoding a primary translation product of 212 amino acids, including an N-terminal 17-amino acid secretion signal sequence. Furthermore, FEM1p contains two potential N-glycosylation sites, and is rich in serine and threonine residues (29%) that could serve as O-glycosyl addition sites. At its C-terminus the protein contains a 22-amino acid sequence with the characteristics of a glycosyl-phosphatidylinositol (GPI) anchor addition signal. A mutant FEM1 protein lacking this GPI anchor addition signal is not retained in the fungal cell wall but released into the culture medium, indicating that in the wild-type protein this sequence functions to anchor the protein to the extracellular matrix. Southern analysis shows that FEM1 is present as a single-copy gene in all formae speciales of F. oxysporum tested and in F. solani. Database searches show that FEM1p homologous sequences are present in other filamentous fungi as well.     

Biologically active domain in somatomedin-binding protein

We have found that human decidua synthesizes a 34K somatomedin-binding protein PP12. Purification of PP12 by immunochemical techniques from human placenta and adjacent membranes has also yielded lower-molecular weight immunoreactive polypeptides designated as PP12B. An individual 21K fragment of somatomedin-binding protein, and a mixture of fragments with molecular weight from 17K to 20K were isolated from this material using high performance liquid chromatography (HPLC). These fragments reacted with antibodies to native PP12 as shown by Western blotting. They all shared the same N-terminal amino acid sequence: Ala-Pro-Trp-Gln-, which is identical with that obtained for PP12. The 21K fragment was shown to bind somatomedin-C, or IGF-I (insulin-like growth factor-I). Since the N-terminal end of the 21K fragment is identical with that of the 34K somatomedin-binding protein, our results suggest that the 21K fragment is the N-terminal part of somatomedin-binding protein, and the somatomedin-binding domain resides in this N-terminal portion.