Lawrence J. Dangott

Membrane guanylate cyclase is a cell-surface receptor with homology to protein kinases

Guanylate cyclase has been strongly implicated as a cell-surface receptor on spermatozoa for a chemotactic peptide1, and on various other cells as a receptor for atrial natriuretic peptides2–4. Resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), the chemotactic peptide released by sea urchin Arbacia punctulata eggs, is specifically crosslinked to A. punctulata spermatozoan guanylate cyclase1. After the binding of the peptide the state of guanylate cyclase phosphorylation modulates enzyme activity1,5,6. We report here that the deduced amino-acid sequence of the spermatozoan membrane form of guanylate cyclase predicts an intrinsic membrane protein of 986 amino acids with an amino-terminal signal sequence. A single transmembrane domain separates the protein into putative extracellular and cytoplasmic-catalytic domains. The cytoplasmic carboxyl-terminal 95 amino acids contain 20% serine, the likely regulatory sites for phosphory-lation. Unexpectedly, the enzyme is homologous to the protein kinase family.

Sm1, a Proteinaceous Elicitor Secreted by the Biocontrol Fungus Trichoderma virens Induces Plant Defense Responses and Systemic Resistance

The soilborne filamentous fungus Trichoderma virens is a biocontrol agent with a well-known ability to produce antibiotics, parasitize pathogenic fungi, and induce systemic resistance in plants. Even though a plant-mediated response has been confirmed as a component of bioprotection by Trichoderma spp., the molecular mechanisms involved remain largely unknown. Here, we report the identification, purification, and characterization of an elicitor secreted by T. virens, a small protein designated Sm1 (small protein 1). Sm1 lacks toxic activity against plants and microbes. Instead, native, purified Sm1 triggers production of reactive oxygen species in monocot and dicot seedlings, rice, and cotton, and induces the expression of defense-related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development under different nutrient conditions and in the presence of a host plant. Using an axenic hydroponic system, we show that SM1 expression and secretion of the protein is significantly higher in the presence of the plant. Pretreatment of cotton cotyledons with Sm1 provided high levels of protection to the foliar pathogen Colletotrichum sp. These results indicate that Sm1 is involved in the induction of resistance by Trichoderma spp. through the activation of plant defense mechanisms.

Maize (Zea mays)‐derived bovine trypsin: characterization of the first large‐scale, commercial protein product from transgenic plants

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal‐derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial‐level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large‐scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal‐derived trypsin in the processing of pharmaceutical proteins.

The catalytic mechanism for aerobic formation of methane by bacteria

Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms. These bacteria apparently catalyse the formation of methane through the cleavage of the highly unreactive carbon–phosphorus bond in methyl phosphonate (MPn), but the biological or terrestrial source of this compound is unclear. However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate and the bacterial C–P lyase complex is known to convert MPn to methane. In addition to MPn, the bacterial C–P lyase complex catalyses C–P bond cleavage of many alkyl phosphonates when the environmental concentration of phosphate is low. PhnJ from the C–P lyase complex catalyses an unprecedented C–P bond cleavage reaction of ribose-1-phosphonate-5-phosphate to methane and ribose-1,2-cyclic-phosphate-5-phosphate. This reaction requires a redox-active [4Fe–4S]-cluster and S-adenosyl-l-methionine, which is reductively cleaved to l-methionine and 5′-deoxyadenosine. Here we show that PhnJ is a novel radical S-adenosyl-l-methionine enzyme that catalyses C–P bond cleavage through the initial formation of a 5′-deoxyadenosyl radical and two protein-based radicals localized at Gly 32 and Cys 272. During this transformation, the pro-R hydrogen from Gly 32 is transferred to the 5′-deoxyadenosyl radical to form 5′-deoxyadenosine and the pro-S hydrogen is transferred to the radical intermediate that ultimately generates methane. A comprehensive reaction mechanism is proposed for cleavage of the C–P bond by the C–P lyase complex that uses a covalent thiophosphate intermediate for methane and phosphate formation.

Residual α2-macroglobulin in fetal calf serum and properties of its complex with thrombin

Summary Active α2-macroglobulin (α2M) has been shown to persist in commercial preparations of fetal bovine serum. Reaction with trypsin and the mitogenic protease thrombin leads to cleavage of the polypeptide chain of α2M, with simultaneous conversion to a fast electrophoretic form. The trapped protease is fully active toward low molecular weight synthetic substrates, but reacts slowly if at all with the protein inhibitor hirudin (M.W. 12,200). Addition of methylamine to fetal bovine α2M leads to covalent incorporation of the amine into the protein, but the subsequent and apparently spontaneous conversion from the slow to the fast electrophoretic form seen with human α2M is not observed. Sedimentation velocity studies of intact and modified fetal bovine α2M show that a small shape change results from reaction with proteases but that, in contrast to the human protein, no shape change results from reaction with methylamine.

Erythrocruorins of Euzonus mucronata treadwell: Evidence for a dimeric annelid extracellular hemoglobin

Abstract 1. 1. The extracellular hemoglobin of the opheliid polychaete Euzonus mucronata is unusual in that it consists of a 3.2 × 10 6 as well as a 6–7 × 10 6 molecular weight hemoglobin at pH 7.O. 2. 2. Electron microscopy of the 3.2 x 10 6 dalton material shows the typical annelid hemoglobin pattern of a two-tiered hexagonal array of submultiples whereas the 6–7 × 10 6 dalton pigment has a four-tiered hexagonal arrangement, a putative dimer of a 3.2 × 10 6 molecular weight material. A few six-tiered trimeric stacks are also present. 3. 3. The dimeric material, which represents 10–15% of the total pigment, is present in unpurified vascular fluid as well as in purified hemoglobin. The constant stoichiometry between the 3.2 × 10 6 dalton monomer and the dimeric pigment under different methods of purification as well as in hemoglobin examined directly from the worm suggests that the dimer is not a preparative artifact. The dimer is not in a state of rapid dissociation-association equilibrium with the monomer nor does the dimer seem to be the result of a ligand-linked association of the 3.2 × 10 6 dalton monomer. 4. 4. The subunit molecular weights of both the monomer and dimer are the same with polypeptide chains of 36,700 ± 800, 31,400 ± 800. 14,400 ± 300. and 12,500 ± 300 as determined by sodium dodecyl sulfate gel electrophoresis. The same molecular weight heterogeneity is present for globin which has been performic acid oxidized, carboxymethylated in 7–8 M guanidine HC1 and 0.1 M in 2-mercaptoethanol, or treated with 8 M urea, 0.1 M in 2-mercaptoethanol. 5. 5. The hemoglobin contains one heme per 22,400 g protein. The amino acid composition of this protein is presented.

Regulation of Phenylalanine Hydroxylase: Conformational Changes Upon Phenylalanine Binding Detected by Hydrogen/Deuterium Exchange and Mass Spectrometry

Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.

Expression and purification of biologically active Trichoderma virens proteinaceous elicitor Sm1 in Pichia pastoris

The beneficial fungus Trichoderma virens secretes a small cysteine-rich protein (Sm1) that induces defense responses in dicot and monocot plants and is a member of the cerato-platanin family. Purification of Sm1 from T. virens results in low protein yield limiting the application of this protein for crop disease protection to small-scale assays. To increase the yield of Sm1, we cloned the sm1 gene in the pPIC9K vector for transformation into the AOX1 locus of Pichia pastoris strain GS115. Transformants of P. pastoris were selected based on the presence of the vector insert as indicated by PCR analysis and the ability to secrete high levels of the rSm1 protein. The optimal incubation period and methanol concentrations for induction were determined for production of rSm1 in shake flasks. One Pichia transformant was estimated to express approximately 55 mg/l of rSm1 after 4 days culture in a 1% final concentration of methanol. The secreted rSm1 was purified by ammonium sulfate precipitation, ion exchange chromatography and gel column chromatography. SDS-PAGE and Western blot analysis revealed that the purified rSm1 expressed in Pichia was recognized by anti-Sm1 polyclonal antibody. The protein sequence was verified by ESI/MS/MS analysis of a tryptic digest of the rSm1. Greater than 90% peptide coverage was obtained and determined to be identical to the predicted sequence. The MALDI/TOF/MS analysis revealed the molecular mass of rSm1 to be 13.1 kDa, which is higher than native Sm1 (12.6 kDa). Edman sequencing of the purified protein revealed an N-terminal extension of six amino acids (EAEAYV). The extension is the result of insufficient activity of the Ste13 protease preventing efficient cleavage of the spacer (EAEA) downstream of the Kex2 cleavage site. Maize (cv. Silver Queen) treated with rSm1 or native Sm1 demonstrated the induction of two defense genes. Enhanced production of this elicitor has implications for the treatment of specialty crops to promote disease resistance.

Regulation of phenylalanine hydroxylase

Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.

Structural studies of a branchiopod crustacean (Lepidurus bilobatus) extracellular hemoglobin. Evidence for oxygen-binding domains.

The extracellular hemoglobin of the notostracan branchiopod Lepidurus bilobatus has an apparent molecular weight of 680,000 and may exist in a dissociation-association equilibrium dependent on pH and ligand state. The pigment contains one heme per 18,000 g protein. However, attempts to dissociate the hemoglobin by harsh denaturing conditions results in a 33-34,000 molecular weight polypeptide chain as well as traces of some 62-64,000 molecular weight material. Limited proteolysis of this hemoglobin with subtilisin produces 14,800 and 16,500 dalton heme-containing polypeptides (domains) which bind oxygen reversibly. These domains, isolated by column chromatography, have a heme content similar to the intact pigment. It is proposed that the intact 34,000 dalton subunit of Lepidurus hemoglobin consists of two linearly linked oxygen binding domains. Oxygen binding properties of the intact hemoglobin show a low oxygen affinity with a slight Bohr effect. In contrast, the isolated domains display a relatively high oxygen affinity and lack a Bohr effect between pH 7.0 and 8.0. It is apparent that the intact 34,000 dalton polypeptide is necessary for the expression of the heterotropic interactions of the native pigment.