Proteoglycans of soluble fraction of mouse mastocytoma.

Proteoglycans have been isolated from a high speed supernatant fraction of a mouse mastocytoma by procedures which should minimize alteration of the native protein-polysaccharide molecule. The methods used include in vivo labeling proteoglycans with 35S-sulfate, 3H-leucine and 3H-lysine, centrifugation of the tumor homogenate at 105,000 g, cetylpyridinium fractionation of the supernatant, and further purification of some of the fractions obtained by DEAE-cellulose column chromatography, gel filtration on Sepharose 4B and cellulose acetate electrophoresis. Two major sulfated proteoglycans were obtained, one containing keratan sulfate-like material (KSP-S), the other a heparin-like polymer (HP-S). The presence in HP-S of a compound similar to heparin was confirmed by its digestibility with flavobacterium heparinase. HP-S contained about 4 per cent protein. Glycine was the predominant amino acid, and serine did not appear to be involved in the peptide-carbohydrate linkage. The proteoglycan present in HP-S appeared to be homogeneous when examined using cellulose acetate electrophoresis. KSP-S was found to contain sialic acid and its protein content was significantly higher than that of HP-S. Glutamic and aspartic acids were the most abundant amino acids in KSP-S.     

Proteoglycans of Microsomal Fraction of Mouse Mastocytoma

Proteoglycans have been isolated from a microsomal fraction of a mouse mastocytoma by procedures which should minimize alteration of the original protein-polysr, ccharide molecule. The methods used include in vivo labeling of sulfate-containing proteoglycans with³⁵S-sulfate, centrifugation of the tumor homogenate at 105, 000 g, solubilization of the microsomal pellet using sodium dodecyl sulfate, cetylpyridinium fractionation, DEAE-cellulose column chromatography and Geon resin electrophoresis. Two major sulfated proteoglycan fractions were obtained. The analytical data obtained were interpreted to indicate that one of these fractions contained keratan sulfate-like material (KSP), the other a heparin-like polymer (HP). KSP was found to contain sialic acid. The protein content of KSP was considerably higher than that of HP. Results of amino acid analysis indicate that glutamic acid and leucine were predominant in KSP, but serine and glycine in HP. Both KSP and HP were found to be homogeneous when examined using acrylamide gel and cellulose acetate electrophoresis, and HP using Geon resin electrophoresis.     

Isolation of an acidic protein from the armadillo submandibular gland

An acidic protein fraction with an apparent molecular weight of 34 000 has been isolated from the Cetavlon-treated, mucin-free supernatant of the armadillo submandibular gland 0.01 M NaCl extract. This purified material, which was obtained in a yield of 0.45%/g wet gland, contains 24 mol % acidic amino acids and 4 mol % basic amino acids. Hexosamines, sialic acid, and neutral sugars represent 7% of the dry sample weight. In polyacrylamide gel and cellulose acetate electrophoresis, a single protein band was observed. The acidic protein fraction is highly reactive with the Lowry phenol reagent, giving a protein value 83% higher than that obtained by summation of its anhydrous amino acids, and is explained by the occurrence of peptide linkages peculiar to this material. The presence of other basophilic components besides mucus glycoproteins within the salivary gland of the armadillo may have physiological significance.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

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.     

Thein vitro activity of the microsomal fraction isolated from the spleen and the lymph nodes after immunization of the donor

The microsomal fraction from the spleen (after perfusion) of immunized rabbits incubated for 20 min at 37° C under usual conditions in the presence of energy sources incorporates¹⁴C-labelled amino acids both into the solubilized (by adding deoxycholate), and into the nonsolubilized part (15%). The cell supernatant incorporates under these conditions the¹⁴C-labelled amino acids into total proteins in the absence of microsomes but in a lower degree. The cell supernatant contains gamma globulin detectable by immunoelectrophoresis. Gamma globulin obtained by specific precipitation of the solubilized microsomal fraction with antigamma-globulin serum had an measurable radioactivity. The precipitate of gamma globulin obtained from the supernatant of the incubation medium in the same manner (after removing the microsomes) had a specific activity twice as high. On separating the microsomal fraction extract and the incubation medium supernatant on DEAE cellulose most fractions show on extinction maximum at 260 nm in the first case and at 280 nm in the second case. The microsomal fraction isolated from the spleen and lymph nodes of immunized pigs-48 and 72 h after revaccination, when incubatedin vitro, incorporated¹⁴C-labelled amino acids into total protein. After ultrasonic disintegration in 0.14m NaCl and filtration through a Sephadex G 25 column it is specifically precipitated with the antigammaglobulin serum. Gamma globulin isolated after incubation of the microsomal fraction had a measurable radioactivity. AntiHSA antibodies determined by adsorption on immunosorbent did not possess significant radioactivity. Only the concentrated supernatant of the incubation medium showed minute radioactivity of 75–94 counts/min /ml. The problem of investigating the formation of nascent specific antibodies on a subcellular levelin vitro during the early period of secondary response to the antigen is discussed, in particular the problem of their detection. An erratum to this article is available at .     

Glycosaminoglycans and proteoglycans of human chondrosarcoma.

The glycosaminoglycans and proteoglycans of a human chondrosarcoma have been studied. Glycosaminoglycans were fractionated and identified by cetylpirdium 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 fractioned 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 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.     

LABELING WITH 14C AMINO ACIDS OF ALBUMIN-LIKE PROTEIN BY RAT LIVER RIBONUCLEOPROTEIN PARTICLES

Ribonucleoprotein particles were prepared by treatment of rat liver microsomes with detergents and high concentrations of KCl. They were active in incorporating ¹⁴C amino acids into protein when incubated with cell sap together with ATP, GTP, and a system to regenerate the triphosphates. The albumin of the incubation mixture, soluble at 105,000 g, and that of the fraction released by ultrasonication of the particles were studied by immunoelectrophoresis in agar gel. When the ribonucleoprotein particles were incubated with cell sap the immunological precipitation lines formed with antiserum to rat serum albumin were highly radioactive as tested by autoradiography. After zone electrophoresis on cellulose acetate, two immunologically reactive albumins were obtained which differed in their electrophoretic mobility from rat serum albumin. Labeled albumin, when purified on DEAE-cellulose columns, retained its radioactivity as tested by autoradiography following immunoelectrophoresis. On cellulose acetate this purified albumin showed an electrophoretic mobility higher than that of rat serum albumin.     

Isolation of a chondroitin sulfate--dermatan sulfate proteoglycan from bovine aorta.

Material containing proteoglycans was extracted from bovine aorta by the dissociative solvent 3.0 m MgCl². The proteoglycan that remained in solution at low ionic strength was purified by isopycnic CsCl centrifugation (?, 1.75 – 1.89 g/ml). From the lower third of the gradient a proteoglycan was isolated which behaved as a homogeneous material when analyzed by the ultracentrifuge and by electrophoresis on cellulose acetate. The proteoglycan contained 12% protein, 21% uronic acid, and 28% hexosamine. Analyses by hyaluronidase digestion and gas-liquid chromatography of the polysaccharide moieties of the proteoglycan showed a composition of 56% chondroitin 6-sulfate, 20% chondroitin 4-sulfate, and 7% dermatan sulfate. A copolymeric structure for the polysaccharide of the proteoglycan is proposed.     

Metabolism of [2-14C] acetate to amino acids and proteins in segments of bean seedling roots

Summary ¹⁴C from [2-¹⁴C] acetate was found to be incorporated into soluble and protein amino acids in substantial amounts by bean root apices. The ¹⁴C was spread through a wide range of amino acids in both these fractions. Glutamic acid was found to be heavily labelled with ¹⁴C in both soluble and protein amino acid fractions. The data are discussed in relation to present ideas on transport and utilization of amino acids in root systems.     

Isolation and characterization of a sialo-glycopeptide from buffalo colostrum

A sialoglycopeptide was isolated from buffalo colostrum in pure form by chromatography on Sephadex G-25 and QAE-Sephadex A-25. This was found to be homogeneous by cellulose acetate membrane electrophoresis and reverse phase HPLC. It consisted of fucose, galactose, mannose,N-acetyl glucosamine andN-acetyl neuraminic acid in the ratio 12341, and aspartic acid, serine, threonine, proline and glutamic acid were the major amino acids. Glycine was identified as the N-terminal amino acid residue. The structure elucidation of the carbohydrate moiety was carried out by methylation analysis, mass spectrometry,¹H-NMR spectroscopy and the probable structure was revealed to be that of a complex biantennary type.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

Studies on the Dissociation of the Soluble Ovomucin by Sonication

The soluble ovomucin obtained from the liquid part of thick white by gel filtration on a Sepharose 4B was an aggregated and polymerized molecule (intrinsic viscosity was 365 ml/g and molecular weight was 8.3 × 10⁶) and it was unable to dissociate the soluble ovomucin into two components without modifications.

Molecular weight and reduced viscosity of the soluble ovomucin decreased markedly with time of sonication. By the sonication for 10 min, it was successful to fractionate it into carbohydrate rich and poor component by density gradient electrophoresis, cellulose acetate electrophoresis and DEAE-cellulose column chromatography.

Concerning carbohydrate and amino acid compositions of two components obtained from the sonicated soluble ovomucin, it was found that the carbohydrate poor component corresponded to the reduced S-component or the reduced α-ovomucin, and the carbohydrate rich component to the reduced F-component or the reduced β-ovomucin.

It was considered that the sonicated soluble ovomucin was an intermediate of the aggregated, polymerized ovomucin (the soluble ovomucin) and the monomeric ovomucin (the sonicated and reduced soluble ovomucin).     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Acid Soluble Peptides in Rat Skeletal Muscle

The acid soluble peptide fraction was prepared from rat skeletal muscle, and the amino acid composition of the fraction was analyzed. The peptide fraction was rich in glutamic acid (or glutamine) and glycine and was poor in branched chain or aromatic amino acids. Since the peptide fraction contained N^τ-methylhistidine, the fraction or at least a part of it was presumed to be composed of intermediate peptides of protein degradation in skeletal muscle. At least 31 spots were detected in the fraction by one dimensional paper electrophoresis.     

The effects of individual amino acids on the incorporation of labelled amino acids into proteins by brain homogenates

Abstract— The effects of phenylalanine and other amino acids on incorporation of several different ¹⁴C-labelled amino acids into cerebral protein were studied in brain homogenates. Excess of some amino acids had a varied effect with different ¹⁴C-labelled amino acids. Of the unlabelled-labelled amino acid combinations tested the maximal inhibition was obtained with the following: (1) phenylalanine, which inhibited the incorporation of [¹⁴C]tyrosine, and (2) leucine, which inhibited incorporation of [¹⁴C]isoleucine. In both cases the inhibition occurred principally in proteins that were recovered in the 800 g and 13,000 g sediments. Only a small degree of inhibition occurred in proteins that sedimented at 100,000 g, and no inhibition occurred in proteins of the 100,000 g supernatant.     

Isolation and Some Properties of Acid Phosphatase-11 from Tomato Leaves

An isozyme of acid phosphatase-1, acid phosphatase-1¹, was purified from the leaves of tomato (Lycopersicon esculentum) to homogeneity and characterized. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate. The gel filtration analysis showed that the native molecule had a relative molecular mass of about 61 kilodaltons (kDa). The relative molecular mass of the subunit on gel electrophoresis with sodium dodecyl sulfate was about 32 kDa, indicating that the native form of the enzyme was a homodimer. It was suggested by periodic acid-Schiff staining on the gel that the enzyme was a glycoprotein. The Km for p-nitrophenylphosphate was 2.9 × 10^?3 m. The enzyme had a pH optimum of 4.5 in 0.15 m potassium acetate buffer with p-nitrophenylphosphate as a substrate. This enzyme was activated by divalent metal ions, such as Zn²⁺, Mg²⁺, and Mn²⁺. The N-terminal amino acids were sequenced after the purified enzyme was treated with pyroglutamylpeptidase. It was suggested that the N-terminal amino acid was pyroglutamate.     

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Summary Biosynthetic preparation of²H- and¹³C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing¹³C- or²H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of²H- and¹³C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Isolation, Characterization, and Heterologous Expression of a Carboxylesterase of Pseudomonas aeruginosa PAO1

We purified to homogeneity an intracellular esterase from the opportunistic pathogen Pseudomonas aeruginosa PAO1. The enzyme hydrolyzes p-nitrophenyl acetate and other acetylated substrates. The N-terminal amino acid sequence was analyzed and 11 residues, SEPLILDAPNA, were determined. The corresponding gene PA3859 was identified in the P. aeruginosa PAO1 genome as the only gene encoding for a protein with this N-terminus. The encoding gene was cloned in Escherichia coli, and the recombinant protein expressed and purified to homogeneity. According to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and analytical gel filtration chromatography, the esterase was found to be a monomer of approximately 24 kDa. The experimentally determined isoelectric point was 5.2 and the optimal enzyme activity was at 55°C and at pH 9.0. The esterase preferentially hydrolyzed short-chain fatty acids. It is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by ethylendiaminotetraacetic acid (EDTA). Native enzyme preparations typically showed a Michaelis constant (K_m) and V_max of 0.43 mM and 12,500 U mg^–1, respectively, using p-nitrophenyl acetate as substrate. Homology-based database searches clearly revealed the presence of the consensus GXSXG signature motif that is present in the serine-dependent acylhydrolase protein family.