Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin.

Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109.     

Phosphate transport by rat intestinal basolateral-membrane vesicles.

The characteristics of phosphate transport across intestinal basolateral membranes of the rat were determined by using enriched preparations in which uphill Na+-dependent D-glucose transport could not be demonstrated, but ATP-dependent Ca2+ transport was present. Phosphate transport was saturable, Na+-dependent and exhibited Michaelis-Menten kinetics. Vmax. was 51.1 +/- 4.2 pmol/10 s per mg of protein and Km was 14 +/- 3.9 microM. The transport process was electroneutral. Tracer-exchange experiments and counter-transport studies confirmed the presence of a Na+-Pi carrier at the basolateral membrane. The presence of inside-positive membrane potential did not enhance phosphate uptake, indicating that the Na+ effect is secondary to the presence of the Na+-Pi carrier rather than an induction of positive membrane potential. The stoichiometry of this carrier at pH 7.4 was 2 Na+:1 phosphate, as shown by direct studies utilizing the static-head method. These studies are the first to determine the presence of a phosphate carrier at the basolateral membrane.     

Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase.

The neutrophil enzyme myeloperoxidase generates hypochlorous acid (HOCl) at sites of inflammation. Glutathione peroxidase is very quickly inactivated by low concentration of HOCl. Inactivation of catalase is also rapid, but requires higher HOCl concentrations and the haem appears to be degraded. Inactivation of bovine CuZn superoxide dismutase is slower. Hence superoxide dismutase should not be easily inactivated by HOCl at sites of inflammation, which may contribute to its effectiveness as an anti-inflammatory agent and in minimizing reperfusion injury.     

Biochemistry of the autolytic processes in Antarctic krill post mortem. Autoproteolysis.

1. Autoproteolysis post mortem was examined at 0 degree C by following the changes in the major classes of krill (Euphausia superba and Euphausia crystallorophias) proteins and by liberation of peptides and free amino acids, and was based on experiments conducted on board expedition vessels in the Antarctic. 2. Primarily salt-soluble proteins were broken down during the first week of incubation, whereas water-soluble and insoluble proteins were degraded to a much smaller extent. The enzymes responsible for the hydrolysis presumably originate primarily from the digestive apparatus of the krill. 3. In general, the individual amino acids were released at rates corresponding to their relative occurrence in the bulk protein of the krill. Alanine was liberated in larger amounts than would be expected from the composition of the krill protein, and was evidently formed also by reactions other than proteolysis. Glutamic acid, and certain amino acids which presumably occur with high frequency adjacent to glumatic acid residues in the krill protein, were liberated only to a limited extent, and accumulated in smaller peptides. 4. During proteolysis, arginine seemed to be converted to some degree into ornithine, and on prolonged incubation conversion of arginine and lysine into their corresponding decarboxylation products, agmatine and cadaverine, appeared to take place.     

A study of the interaction between cartilage proteoglycan and link protein.

The interaction between proteoglycan and link protein extracted from bovine articular cartilage (15-18-month-old animals) was investigated in 0.5 M-guanidinium chloride. The proteoglycans, radiolabelled as the aggregate (A1 fraction), were fractionated by two 'dissociative' density-gradient centrifugations (A1D1D1) followed by a rate-zonal centrifugation (S1) to yield an A1D1D1S1 preparation. At least 65% of these proteoglycans were able to bind to hyaluronate, but only 52% were able to bind to link protein as assessed by chromatography on Sepharose CL-2B. Over 80% of the [3H]link-protein preparation, radiolabelled as the aggregate, was able to interact with proteoglycan as assessed by chromatography on Sepharose CL-4B. Equilibrium-boundary-centrifugation studies performed at low link-protein concentrations (2.42 x 10(-9) M-5.93 x 10(-8) M) were analysed by Scatchard-type plots and indicated a Kd of 1.5 x 10(-8) M and a stoichiometry, n = 0.56, i.e. approx. 56% of those proteoglycans capable of binding to link protein had a strong site for link protein if a 1:1 stoichiometry were assumed. However, experiments performed at higher link-protein concentrations (3.5 x 10(-7) M and 8 x 10(-7) M) yielded stoichiometry values which were link-protein-concentration-dependent. Non-equilibrium binding studies using chromatography on Sepharose CL-2B and rate-zonal centrifugation yielded apparent stoichiometries between 0.6 and 7.5 link-protein molecules per proteoglycan monomer as a function of increasing link-protein concentration. It was concluded that a proportion of the proteoglycan molecules had a strong site for binding a single link protein (Kd 1.5 x 10(-8) M) and that at high link-protein concentrations a weaker, open-ended, process of link-protein self-association nucleated upon the strong link-protein-proteoglycan complex occurred. Hyaluronate oligosaccharides appeared to abolish a proportion of this self-association (as observed by Bonnet, Dunham & Hardingham [(1985) Biochem. J. 228, 77-85] in a study of link-protein-hyaluronate-oligosaccharide interactions) so as to leave a link protein:proteoglycan stoichiometry of 2. It is not clear whether this second link-protein molecule binds directly to the proteoglycan or to the first link protein.     

Biochemical characterization of individual normal, floppy and rheumatic human mitral valves.

Human mitral valves (32 floppy and 17 rheumatic) obtained at surgery were analysed and compared with 35 normal (autopsy) valves. Total amounts of collagen, proteoglycan and elastin were increased approx. 3-fold in floppy and rheumatic valves. The water content of rheumatic cusps was lower than normal. The most significant changes in floppy valves were the 59% increase in mean value of the proteoglycan content, a large increase in the ease of extractability of proteoglycans from 26.7 to 57.2% of the total and a 62% increase in mean value of the elastin content in the anterior cusps. Normal human mitral valve cusps contained a mean proportion of 29.3 (and chordae 26.6) type III collagen (as % of total types III + I collagen), the values increasing significantly to 33.2 and 36.3% respectively in chronic rheumatic disease. The ratio observed in floppy valves depended on the extent of secondary surface fibrosis, which could be demonstrated histologically; in valve cusps with considerable secondary fibrosis, the percentage of type III increased significantly (to 34.4%), whereas it decreased significantly (to 25.2%) when fibrosis was negligible. It is concluded that the ratio of collagen types in floppy valves reflects the extent of secondary fibrosis rather than the pathogenesis of the disrupted collagen in the central core of the valve.     

Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.     

Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane.

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the 'cold' synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.     

Treatment of cartilage proteoglycan aggregate with hydrogen peroxide. Relationship between observed degradation products and those that occur naturally during aging.

The effects of treatment of purified neonatal human articular-cartilage proteoglycan aggregate with H2O2 were studied. (1) Exposure of proteoglycan aggregate to H2O2 resulted in depolymerization of the aggregate and modification of the core protein of both the proteoglycan subunits and the link proteins. (2) Treatment of the proteoglycan aggregate with H2O2 rendered the proteoglycan subunits unable to interact with hyaluronic acid, with minimal change in their hydrodynamic size. (3) Specific cleavages of the neonatal link proteins occurred. The order in which the major products were generated and their electrophoretic mobilities resembled the pattern observed during human aging. (4) The proteolytic changes in the link proteins were inhibited in the presence of transition-metal-ion chelators, thiourea or tetramethylurea, suggesting that generation of hydroxyl radicals from H2O2 by trace transition-metal ions via a site-specific Fenton reaction may be responsible for the selective cleavages observed. (5) Cleavage of the link proteins in proteoglycan aggregates by H2O2 was shown to have a limited effect on the susceptibility of these proteins to cleavage by trypsin. (6) The relationship between these changes and those observed in cartilage during human aging suggests that some of the age-related changes in the structure of human cartilage proteoglycan aggregate may be the result of radical-mediated damage.     

Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.