Biochemical studies of soluble atrial natriuretic peptide (ANP) receptors from rat olfactory bulb and vascular smooth muscle cells

1. Aim. The biochemical characteristics of atrial natriuretic peptide receptors (ANP-R) derived from rat vascular smooth muscle (A-10 cell line) and central nervous system (CNS; olfactory bulb) tissue were compared. 2. Method and Results. ANP-Rs from each source were solubilized with 40 to 65% efficiency utilizing the nonionic detergent Lubrol-PX. Upon solubilization, the ANP-R from each source maintained the ability to bind 125I-ANP (99-126) with a high affinity; Scatchard analysis indicated that the VSMC ANP-R displayed a Kd for the radioligand of approximately 10 pM, whereas the olfactory receptor possessed a Kd of about 165 pM. The Bmax values for the soluble VSMC and olfactory ANP-Rs were 285 and 30 fmol/mg protein, respectively. Competition binding studies indicated that the VSMC ANP-R bound ANP(99-126), ANP(103-126), and ANP(103-123) with similar affinities, whereas the olfactory ANP-R was much more sensitive to changes in the COOH-terminal structure of the competing peptide. The soluble ANP-Rs from VSMC and olfactory were chromatographically indistinguishable on phenyl-, DEAE-, and wheat germ agglutinin-agarose columns. However, the ANP-Rs could be distinguished using GTP-agarose; the olfactory ANP-R was capable of binding to the resin, whereas the VSMC ANP-R was not. 3. Conclusions. Coupled with other studies, these data suggest that the A10 VSMC ANP-R observed in this study may not be coupled to guanylate cyclase and may represent a receptor serving a clearance function, whereas a significant proportion of the olfactory CNS ANP-R appears to be associated with GTP-binding proteins, likely particulate guanylate cyclase, and probably represents a coupled form of the receptor.     

Nature of forces stabilizing the transmembrane protein bacteriorhodopsin in purple membrane

Analysis of the far-ultraviolet solution and the oriented-film circular dichroic (CD) spectra of the purple membrane (PM) has indicated that the α-helical segments of its sole protein bacteriorhodopsin (bR) can undergo a significant tilting from the normal to the membrane plane during light-dependent hydroxylamine-mediated bleaching of the bR. However, this drastic change in tertiary structure is free of any observable secondary structural changes. This phenomenon can provide an excellent means for studying the relative contributions of forces responsible for the stability of this transmembrane protein within the membrane bilayer. Perturbation of the PM by varying degrees of papain digestion (resulting in changes in the bR ranging from only an elimination of the long COOH-terminal tail to the additional eliminations of the short NH₂-terminal tail and a number of linkage amino acids between the helical segments of the bR) and by chemical cross-linking with dimethyl adipimidate (resulting primarily in the formation of intramolecular cross-links) resulted in a significant increase in this bleaching-induced tilting in all cases except the one in which only the COOH-tail was eliminated. The most severe perturbation (2-wk papain digestion) increased the net tilt angle per segment from 24 to 39° with no indication of any secondary structural changes. Although these perturbations drastically reduced the structural stability of the bR to bleaching, they caused virtually no observable changes in the intramolecular structure of the bR or the supramolecular structure of the PM based on analysis of extensive absorption, linear dichroic, and CD spectra. In addition, study of the bleaching rates for the perturbed PM samples indicated that a linear correlation exists between the calculated initial bleaching rates and the net tilt angles.

Considering the forces generally assumed to account for the stability of transmembrane proteins in membranes, (a) intersegmental hydrogen bonding and electrostatic interactions, (b) electrostatic interactions between hydrophilic polypeptide segments extending outside the bilayer and the many charged lipid heads of the bilayer, and (c) hydrophobic interactions, it is clear that the results of the bleaching experiments eliminate all but perhaps the last as contributing significantly to the bR stability in the PM. Furthermore, they provide more compelling evidence than previously available that the bR is capable of undergoing relatively large retinyldiene-controlled tertiary structural changes and that the chromophoric retinal serves as the most important factor in the native bR structural stability. This dynamic view of the bR bears directly on models proposed for bR function, favoring those in which protein structural metastability, rather than rigidity, is an essential factor. The proteinquake or deformation wave model proposed by this laboratory falls into this category.

     

ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma-35S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines.     

Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new species of Crepidostomum (Allocreadiidae: Digenea) from northeastern Finland,with comments on its possible origin

Crepidostomum wikgreni n. sp. is described from the gall-bladder and intestine of the whitefish Coregonus acronius in Lake Yli-Kitka in NE Finland. It is morphologically similar to Crepidostomum farionis, with which it occurs sympatrically and sometimes concurrently; but it differs in that the eggs are much larger, i.e. 96±6.5 m mean-length, as opposed to 71±4.7 m mean-length for C. farionis in the same host and locality.It is suggested that the new species has arisen from C. farionis after deglaciation and since c. 8,400 BP, at which time the waters of the Lake Kitka System were isolated from those in the rest of Finland and flowed eastwards into the White Sea Basin. The isolation of the White Sea Basin appears to have been maintained by watersheds running north-south close to the Fenno-Soviet border and east-west through central Karelia. It is further suggested that C. farionis currently occurring in Lake Yli-Kitka is a recent re-introduction brought about by the translocation of fish-stocks.     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones

N-Acetylgalactosamine-4-sulphatase (EC 3.1.6.1, G4S) is composed of a 57 kDa species in human liver that dissociates into 43 kDa and 8 kDa subunits under reducing conditions and, when deficient, causes the lysosomal storage disorder, mucopolysaccharidosis type VI. We isolated genomic clones containing the G4S first exon, including the leader peptide and the amino terminus of the 43 kDa polypeptide. Amino-terminal amino acid sequences of the 43 kDa and 8 kDa subunits indicated that the 8 kDa component is linked to the 43 kDa polypeptide by a single disulphide bond, does not contain the mannose-6-phosphate lysosomal targeting signal and is at the carboxyl terminus of G4S.