The composition and protein metabolism in the immature rabbit intervertebral disc

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.     

Effects of pregnancy on bone matrix proteins in Wistar rats

Using an EDTA extraction procedure, bones from pregnant Wistar rats were analyzed for their content of collagen and non-collagenous components (sialoprotein, proteoglycan and carbohydrate). The bone matrix size was found to be smaller in pregnant rats than in normal rats (19.5% vs 17.5% of the dry weight bone). The EDTA extractability of the bone protein from pregnant rats was higher than that from controls (2.6% vs 1.9% dry weight bone). EDTA extracts from pregnant rats contained higher amounts of soluble collagen (1.6% vs 0.5% of dry weight tissue) and lower amounts of non-collagenous components (1.65% vs 2.23% for hexoses, 2.38% vs 3.95% for sialic acid and 1.24% vs 1.73% for uronic acid). In bone matrix, collagen content was lower in the pregnant rats (9.45% vs 10.6%). Similarly, the amounts of non-collagenous components were slightly decreased in the bone matrix from the pregnant rats. The respective values were: 0.91% vs 0.93% for hexoses, 0.45% vs 0.52% for sialic acid and 0.39% vs 0.50% for uronic acid. These results suggest that in pregnancy collagen and non-collagenous protein content in bone is decreased while the total mineral content is increased.     

A comparison of methods for the isolation and fractionation of reticulocyte ribosomes

1. Polysomes, ribosomes and pH5 enzymes were isolated from rabbit reticulocytes by acidifying the post-mitochondrial supernatant to pH6.0 to precipitate all ribonucleoprotein particles and about half the pH5 enzymes; the precipitate was redissolved in buffer, pH7.6, and fractionated by zone centrifuging. 2. The isolation of polysome-rich and ribosome-rich fractions from the post-mitochondrial supernatant was also examined. 3. Studies of the stability of polysomes revealed that dissociation into sub-units occurred when both bound and free Mg(2+) was chelated by EDTA or when the pH was increased above pH8.8.     

The specificity of proteinases from Streptomyces griseus (pronase)

Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A(2) contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-l-arginine and hippuryl-l-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.     

Axonal transport of proteoglycans to the goldfish optic tectum

The study addressed the question of whether³⁵SO₄ labeled molecules that the have been delivered to the goldfish optic nerve terminals by rapid axonal transport include soluble proteoglycans. For analysis, tectal homogenates were subfractionated into a souluble fraction (soluble after centrifugation at 105,000g), a lysis fraction (soluble after treatment with hypotonic buffer followed by centrifugation at 105,000g) and a final 105,000g pellet fraction. The soluble fraction contained 25.7% of incorporated radioactivity and upon DEAE chromatographys was resolved into a fraction of sulfated glycoproteins eluting at 0–0.32 M NaCl and containing 39.5% of total soluble label and a fraction eluting at 0.32–0.60 M NaCl containing 53.9% of soluble label. This latter fraction was included on columns of Sepharose CL-6B with or without 4 M guanidine and after pronase digestion was found to have 51% of its radioactivity contained in the glycosaminoglycans (GAGs) heparan sulfate and chondroitin (4 or 6) sulfate in the ratio of 70% to 30%. Mobility of both intact proteoglycans and constituent GAGs on Sepharose CL-6B indicated a size distribution that is smaller than has been observed for proteoglycans and GAGs from cultured neuronal cell lines. Similar analysis of lysis fraction, containing 11.5% of incorporated³⁵SO₄, showed a mixture of heparan sulfate and chondroitin sulfate containing proteoglycans, apparent free heparan sulfate and few, if any, sulfated glycoproteins. Overall, the result support the hypothesis that soluble proteoglycans are among the molecules axonally transported in the visual system.     

Release of Chromaffin Granule Glycoproteins and Proteoglycans from Potassium-Stimulated PC12 Pheochromocytoma Cells

Abstract: Cultured PC12 pheochromocytoma cells were labeled with [³H]gIucosamine, and the glycoproteins and proteoglycans released following potassium-induced depolarization were fractionated and characterized. Exposure of PC12 cells for 20 min to a high concentration of potassium (51.5 mM in Krebs-Ringers-HEPES buffer) results in an approximately sixfold increase in the release of labeled glycoproteins and proteoglycans, compared to incubation in physiological levels of potassium (6 mM ). The released complex carbohydrates include chromogranins, dopamine β-hydroxylase, and two chondroitin sulfate/heparan sulfate proteoglycan fractions, which together account for 7.4% of the soluble cell radioactivity. The chromogranins contained galactosyl(β l ± 3 )N-ace tylgalactosamine, as well as several mono- and disialyl O -glycosidically-linked oligosaccharides, and the tetra-saccharide AcNeu(α2 ± 3)Gal(β l ± 3)[AcNeu(α2 ± (6)] GalNAcol, obtained by alkaline borohydride treatment of the chromogranin glycopeptides, accounted for almost half of the total chromogranin labeling. The proteoglycan fractions varied in their relative proportions of chondroitin sulfate (23–68%), heparan sulfate (16–23%), and glycoprotein oligosaccharides (16–54%), which are of the triand tetraantennary and O -glycosidic types. As previously found in the case of proteoglycans from bovine chromaffin granules, the more acidic species has a considerably higher proportion of carbohydrate in the form of sulfated glycosaminoglycans.     

Preparation and properties of 5′-nucleotidases from bovine milk fat globule membranes

The 5′-nucleotidase (5′-ribonucleoside phosphohydrolase, EC 3.1.3.5) from bovine milk fat globule membranes was partially purified. Two separate peaks of activity were obtained from a Sepharose column and the two fractions, designated V and VI in order of elution, were collected and characterized separately. Both V and VI exhibited pH optima between 7.0 and 7.5 for AMP, GMP and CMP in the absence of metal ions. In the presence of Mg²⁺, a second pH optimum at 10.0 was observed with both fractions. Low concentrations of MnCl₂ activated Fraction V but not Fraction VI. HgCl₂ was a potent inhibitor of both fractions. The relative rates of hydrolysis of various 5′-mononucleotides differed comparing the two fractions. Optimum temperature for P_i release was 69 °C for both fractions. Activation energies were 10 400 cal/mole and 9600 cal/mole for Fractions V and VI, respectively. For V, calculated K_m values for AMP, GMP and CMP were 0.94, 2.5 and 1.16 mM, respectively. Calculated K_m values for Fraction VI for AMP, GMP and CMP were 5.0, 3.95 and 1.73 mM, respectively. ATP was a competitive inhibitor of AMP hydrolysis by Fraction V and a noncompetitive inhibitor of AMP hydrolysis by Fraction VI. Both fractions contained chloroform-methanol-extractable phospholipid. The phospholipid distribution pattern of Fraction VI was similar to that of milk fat globule membranes. Fraction V contained only sphingomyelin and phosphatidylcholine. It is proposed that milk fat globule membranes contain two separate 5′-nucleotidases.     

Human transfer factor: fractionation and biologic activity.

Human transfer factor (TF) was fractionated by exclusion chromatography and the fractions were tested for biologic activity in vivo and in vitro. Specific TF activity in vivo was found to reside in the major UV-absorbing peak (Fraction III). Fraction III eluted at 2.7 X V(O) and transferred tuberculin, candida, or KLH-reactivity to previously negative recipients. Fraction III from nonreactive donors was ineffective. When the fractions were tested in vitro, we found that both the mitogenic activity of whole TF and the suppressive activity to mitogen activation when present in TF was found in Fraction I. Fraction III contained components responsible for augmentation of PHA and PWM responses. In addition, Fraction III contained the component responsible for antigen-dependent augmentation of lymphocyte transformation. Fraction IV was suppressive to antigen-induced lymphocyte transformation. These data suggest that TF preparations contain components which can affect immune reactions in both specific and nonspecific ways.     

Apurinic DNA endonucleases from Drosophila melanogaster embryos

Summary Apurinic DNA endonuclease activity from Drosophila melanogaster embryos was resolved into two separable forms by phosphocellulose chromatography, one which flowed through the column (Fraction I) and the other which was retained and eluted at approximately 200 mM potassium phosphate (Fraction II). Both fractions, purified further by glycerol gradient sedimentation, were found to introduce nicks into DNA that were specific for and equal in number to the alkali-labile sites in depurinated DNA. They had similar apparent K_m values for apurinic sites (0.7 nM apurinic sites for Fraction I and 0.8 nM for Fraction II), but differed with respect to optimal pH, Mg⁺⁺ requirement and sensitivity to EDTA.     

Proteoglycans of guinea-pig coastal cartilage. Fractionation and characterization.

Proteoglycans were extracted, in a yield of about 90%, from costal cartilage of young, growing guinea-pigs. Three solvents were used in sequence: 0.4 M guanidine - HCl, pH 5.8, 4 M guanidine - HCl, pH 5.8, and 4 M guanidine - HCl/0.1 M EDTA, pH 5.8. The proteoglycans were purified and fractionated by cesium chloride density gradient ultracentrifugation under associative and dissociative conditions. Gel chromatography on Sepharose 2 B of proteoglycan fractions from associative centrifugations showed the presence of both aggregated and monomer proteoglycans. The ratio of aggregates to monomers was higher in the second extract than in the other two extracts. Dissociative gradient centrifugation gave a similar distribution for proteoglycans from all three extracts. Thus, with decreasing buoyant density there were decreasing ratios of polysaccharide to protein, and of chondroitin sulfate to keratan sulfate. In addition, there was with decreasing density an increasing ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. Amino acid analyses of dissociative fractions were inaccordance with previously published results. On comparing proteoglycan monomers of the three extracts, significant differences were found. Proteoglycans, extracted at low ionic strength, contained lower proportions of protein, keratan sulfate, chondroitin 6-sulfate and basic amino acids than those of the second extract. The proteoglycans of the third extract also differed from those of the other extracts. The results indicate that the proteoglycans of guinea-pig costal cartilage exist as a very polydisperse and heterogenous population of molecules, exhibiting variations in aggregation capacity, molecular size, composition of protein core, degree of substitution of the protein core, as well as variability in the type of polysaccharides substituted.     

Identification of organic phosphorus covalently bound to collagen and non-collagenous proteins of chicken-bone matrix. The presence of O-phosphoserine and O-phosphothreonine in non-collagenous proteins, and their absence from phosphorylated collagen

Non-collagenous phosphoproteins, almost all of which can be extracted in EDTA at neutral pH in the presence of proteinase inhibitors, are identified in the matrix of chicken bone, and are therefore not covalently bound to collagen. Similarly, all the peptides containing gamma-carboxyglutamic acid are present in the EDTA extract and none in the insoluble residue, confirming that none is covalently linked to chicken bone collagen. However, organic phosphorus is also found to be present in chicken bone collagen, principally in the alpha2-chains. Of the total protein-bound organic phosphorus present in chicken bone matrix, approx. 80% is associated with the non-collagenous proteins and 20% with collagen. The soluble non-collagenous proteins contain both O-phosphoserine and O-phosphothreonine and these account for essentially of their organic phosphorus content. In contrast, collagen contains neither O-phosphoserine nor O-phosphothreonine. Indeed, no phosphorylated hydroxy amino acid, phosphoamidated amino acid or phosphorylated sugar could be identified in purified components of collagen, which contain approximately four to five atoms of organic phosphorus per molecule of collagen. Peptides containing organic phosphorus were isolated from partial acid hydrolysates and enzymic digests of purified collagen components, which contain an as-yet-unidentified cationic amino acid. These data, the very high concentrations of glutamic acid in the phosphorylated peptides, and the pH-stability of the organic phosphorus moiety in intact collagen chains strongly suggest that at least part of the organic phosphorus in collagen is present as phosphorylated glutamic acid. This would indicate that the two major chemically different protein fractions in chicken bone matrix that contain organic phosphorus may represent two distinct metabolic pools of organic phosphorus under separate biological control.     

Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15–18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2–9% (w/w) of the total sulfated glycoprotein.The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.     

Peptide Mapping Studies of the Chromogranins and of Two Chromaffin Granule Proteoglycans

Abstract: The chromogranins, a family of related acidic glycoproteins, and two chondroitin sulfate/dermatan sulfate proteoglycans were isolated from the soluble contents of bovine adrenal chromaffin granules by chromatography on DEAE-cellulose. These chromaffin granule matrix glycoconjugates were treated with trypsin, and the resulting peptides were fractionated by HPLC. The two proteoglycans, which differ in their concentration of glycosaminogly-cans and glycoprotein oligosaccharides, yielded almost identical peptide patterns and would both appear to have the same protein moiety. The peptide profile of the proteoglycans differs, however, from that of the chromogranins, which they closely resemble in terms of amino acid composition. The various chromogranin fractions obtained by gel filtration were also found to have significant differences in the chromatographic patterns of their tryptic peptides.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa.

Sulfated glycoproteins were extracted and purified from porcine stomach mucous scraping. Four sulfated glycoprotein fractions were separated and subsequently purified. These compounds always accompanied the apparent peptic inhibitory activity and consisted of 15-18% (w/w) protein. The carbohydrate portions contained an equimolar ratio of galactose and hexosamine (mainly glucosamine), together with lesser amounts of fucose and sialic acid. The sulfate content of the above fractions was 2-9% (w/w) of the total sulfated glycoprotein. The mode of inhibition of the sulfated glycoproteins to peptic activity was investigated and suggested that there was binding of the sulfated glycoproteins to the substrate of pepsin, making the substrate resistant to peptic activity. The sulfated glycoproteins neither bound pepsin at pH 1.8 nor inhibited the hydrolysis of a synthetic dipeptide substrate of pepsin. Desulfation of the sulfated glycoproteins resulted in the loss of both the inhibitory activity and the precipitate formation. The precipitation curve for sulfated glycoprotein and porcine serum albumin showed that both bound in varying proportions and suggests that both components are multivalent in this precipitate formation.     

Isolation and partial characterization of soluble alfalfa proteins

Soluble proteins, representing ⋍18% of total plant protein, were extracted from freshly harvested alfalfa. Two fractions based on solubility in one-half saturated (NH₄)₂SO₄—Fraction G (insoluble) and Fraction A (soluble)—were obtained, representing two-thirds and one-third of the extracted protein, respectively. Fraction G contained less hexose-like and more lipid substances than Fraction A which was characterized by an unusually high ash content. Gel electrophoretic patterns revealed greater heterogeneity in Fraction A than in Fraction G, whereas the reverse situation was evident in sedimentation-velocity patterns. The three sedimenting boundaries apparent in Fraction G had S_20,app values of 29.2 (4%), 17.4 (61%) and 3.2 (35%). The S_20,app 3.2 boundary corresponded to the single boundary observed for Fraction A. Moving-boundary electrophoresis revealed a single boundary for each fraction, possessing mobilities of −6.5 (Fraction G) and −7.5 (Fraction A) Tiselius units, respectively.     

Biosynthesis of dimethyl selenide from sodium selenite in rat liver and kidney cell-free systems

A pathway for the synthesis of dimethyl seledine from sodium selenite was studied in rat liver and kidney fractions under anaerobic conditions in the presence of GSH, a NADPH-generating system, and S-adenosylmethionine. Chromatography of liver or kidney soluble fraction on Sephadex G-75 yielded a Fraction C (30 000 molecular weight) which synthesized dimethyl selenide, but at a low rate. Addition of proteins eluting at the void volume (Fraction A) to Fraction C restored full activity. Fractionation of Fraction A on DEAE-cellulose revealed that its ability to stimulate Fraction C was associated with two fractions, one containing glutathione reductase and the other a NADPH-dependent disulfide reductase. It was concluded that Fraction C contains a methyltransferase acting on small amounts of hydrogen selenide produced non-enzymically by the reaction of selenite with GSH, and that stimulation by Fraction A results partly from the NADPH-linked formation of hydrogen selenide catalyzed by glutathione reductase present in Fraction A. Washed liver microsomal fraction incubated with selenite plus 20 mM GSH also synthesized dimethyl selenide, but addition of soluble fraction stimulated activity. A synergistic effect was obtained when liver soluble fraction was added to microsomal fraction in the presence of a physiological level of GSH (2 mM), whereas at 20 mM GSH the effect was merely additive. The microsomal component of the liver system was labile, had maximal activity around pH 7.5, and was exceedingly sensitive to NaAsO₂ (93% inhibition by 10^?6 M arsenite in the presence of a 20 000-fold excess of GSH). The microsomal activity apparently results from a Se-methyltransferase, possibly a dithiol protein, that methylates hydrogen selenide produced enzymically by the soluble fraction or non-enzymically when a sufficiently high concentration of GSH is used.     

Purification and primary sequences of the major arginine-containing antifreeze glycopeptides from the fish Eleginus gracilis

An Arg-containing antifreeze glycoprotein from the polar fish Eleginus gracilis was isolated, and the major components were purified to homogeneity. The general protocol for purification was chromatography of serum on DEAE-cellulose, followed by chromatography on a cation exchanger. DEAE-cellulose chromatography resulted in two fractions, A and B. Fraction A contained most of the antifreeze glycoprotein found in E. gracilis (approximately 80% by weight) and consisted of 13 distinct components. Unlike antifreeze glycoproteins from other previously studied polar fish, Fraction A contained both low and high molecular weight antifreeze glycoprotein components. The two major components of Fraction A were sequenced and compared with the sequence of antifreeze glycoproteins 7 and 8 from both Boreogadus saida and Pagothenia borchgrevinki. The antifreeze glycoproteins from E. gracilis were shown to have a similar composition to those previously studied, except for an additional Ala-Arg dipeptide at the carbon terminal in the major components of Fraction A and the position of Pro in the low molecular weight components. The activity of E. gracilis antifreeze glycoproteins is the subject of a companion article (Burcham, T. S., Osuga, D. T., Yeh, Y., and Feeney, R. E. (1986) J. Biol. Chem. 261, 6390-6397).     

Glycosaminoglycans and proteoglycans of human chondrosarcoma

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpyridium chloride (CPC) cellulose chromatography, ECTEOLA cellulose ion-exchange chromatography and electrophoresis on cellulose acetate. Proteoglycans were extracted by low ionic strength solutions and by 4 M guanidinium chloride and fractionated by equilibrium density-gradient centrifugation and gel chromatography on Sepharose 2B. The tumour matrix contained both the 4- and 6-sulphate isomers of chondroitin sulphate and a high concentration (12% of hexosamine) of hyaluronic acid. Proteoglycans were poor in carbohydrate moieties and a proportion were capable of aggregation. Amino acid analysis of the fractionated proteoglycans suggested the presence of a single protein core. A substance with the characteristic amino acid composition of glycoprotein link was recovered from the top of the dissociative density gradient.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.