In vitro cartilage formation: effects of 6-diazo-5-oxo-L-norleucine (DON) on glycosaminoglycan and collagen synthesis.

To determine the relationships between glycosaminoglycan (GAG) synthesis and type-specific collagen synthesis, we have investigated mouse limbs cultured in the presence of antiglutamine DON (6-diazo-5-oxo-l-norleucine). When compared to control limbs, ultrastructural examination of the DON-treated limbs shows that newly formed cartilage lacks matrix granules and the collagen fibrils have an altered morphology. Using [³⁵S]sulfate as a precursor, we have found that DON (5 μg/ml) suppresses chondroitin sulfate synthesis to less than 15% of the control level. We have also examined the collagen synthesized in equivalent limbs labeled with [³H]proline. The α-chain patterns from CM-cellulose chromatography were very similar for control and experimental limbs (α1:α2 ～ 7), suggesting that both (α1)₃- and (α1)₂α2-type molecules were being produced. The (α1)₃ molecules in both cases were identified as type II collagen by fractional salt separation and cyanogen bromide peptide mapping on CM-cellulose columns. We conclude that (1) the synthesis of type II collagen can be dissociated from the production of GAG, and (2) environmental influences can be involved in controlling the fibrillogenesis of collagen.     

Some changes in the reactivity of enzymes resulting from their chemical attachment to water-insoluble derivatives of cellulose

1. Purified ficin was chemically attached to CM-cellulose, and partially purified ATP–creatine phosphotransferase was chemically attached to both CM-cellulose and p-aminobenzylcellulose. 2. The apparent K_m with respect to ATP and Mg²⁺ of ATP–creatine phosphotransferase was observed to increase about tenfold on attachment of the enzyme to CM-cellulose, and to increase by only 23% on its attachment to p-aminobenzylcellulose. 3. The reactivity of both ficin and ATP–creatine phosphotransferase with 5,5′-dithiobis-(2-nitrobenzoic acid) was observed to decrease on chemical attachment of these enzymes to water-insoluble derivatives of cellulose. With derivatives prepared from CM-cellulose, the extent of the reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) was dependent on ionic strength, but with similar derivatives prepared from p-aminobenzylcellulose the extent of this reaction was independent of ionic strength. 4. The effect of diffusion and electrostatic interaction of charged enzyme substrates and charged enzyme supports on the apparent K_m of a water-insoluble derivative of an enzyme is discussed. An equation is derived that satisfactorily describes the observed effects of these factors on the apparent K_m.     

Isolation and amino acid sequence of a short-chain neurotoxin from an Australian elapid snake, Pseudechis australis.

A short-chain neurotoxin Pseudechis australis a (toxin Pa a) was isolated from the venom of an Australian elapid snake Pseudechis australis (king brown snake) by sequential chromatography on CM-cellulose, Sephadex G-50 and CM-cellulose columns. Toxin Pa a has an LD50 (intravenous) value of 76 micrograms/kg body wt. in mice and consists of 62 amino acid residues. The amino acid sequence of Pa a shows considerable homology with those of short-chain neurotoxins of elapid snakes, especially of true sea snakes.     

Isolation of the alanine carrier from the membranes of a thermophilic bacterium and its reconstitution into vesicles capable of transport

A carrier protein mediatine alanine transport was purified from the membranes of the thermophilic bacterium PS3, by ion exchange chromatography in the presence of both Triton X-100 and urea. The alanine carrier was recovered in the nonadsorbed fraction from either DEAE-or CM-cellulose columns, suggesting that its isoelectric point was in the neutral pH region. The final preparation contained virtually no electron transfer components, ATPase, or NADH dehydrogenase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the final preparation consisted of two major protein components with molecular weights of 36,000 and 9,400. Active transport of alanine after incorporation of the alanine carrier into reconstituted proteoliposomes was driven not only by an artificial membrane potential generated by potassium ion diffusion via valinomycin but also by mitochondrial cytochrome oxidase incorporated into the same liposomes and supplemented with both cytochrome c and ascorbic acid. The membrane-integrated portion (TF₀) of the ATPase complex uncoupled alanine transport by conducting protons across the membrane.     

Purification and Some Properties of Cell Wall-bound Invertases from Sugar Beet Seedlings and Aged Slices of Mature Roots

Cell wall-bound invertases (EC 3.2.1.26) from both sugar beet seedlings and aged slices of mature roots were purified to homogeneity separately with CM-cellulose chromatography and Bio-Gel P-150 gel filtrations. The enzymes behaved similarly throughout the purification procedures. The purified enzymes are identical as characterized by specific activity, gel electrophoretic mobility, K_m for sucrose and raffinose (1.33 and 4.0 millimolar, respectively), mobility on Bio-Gel P-150 (molecular weight 28,000), optimum pH (4.6 to 5.0), optimum temperature, and dependence on NaCl concentration for insolubilization by DNA. The results suggest that the enzymes may be encoded for by the same structural gene.     

Lipoxygenase from Medicago sativa: Purification on hydroxyapatite

Three lipid-oxidizing fractions obtained from an alfalfa extract by stepwise elution from a CM-cellulose column were further purified on hydroxyapatite columns. Although substantial separation between lipoxygenase and peroxidase activities was achieved, only one of the protein fractions migrated as a single band on polyacrylamide gel electrophoresis and exhibited the properties of pure lipoxygenase,     

Catalase from larvae of the camel tick Hyalomma dromedarii

Catalase plays a major role in protecting cells against toxic reactive oxygen species. Here, Catalase was purified from larvae of the camel tick Hyalomma dromedarii and designated TLCAT. It was purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose, Sephacryl S-300 and CM-cellulose columns. Gel filtration and SDS-PAGE of the purified TLCAT indicated that the protein has a native molecular weight of 120 kDa and is most likely a homodimer with a subunit of approximately 60 kDa. The Km value of TLCAT is 12 mM H₂O₂ and displayed its optimum activity at pH 7.2. CaCl₂, MgCl₂, MnCl₂ and NiCl₂ increased the activity of TLCAT, while FeCl_2, CoCl₂, CuCl₂ and ZnCl₂ inhibited the activity of TLCAT. Sodium azide inhibited TLCAT competitively with a Ki value of 0.28 mM. The presence of TLCAT in cells may play a role in protecting H. dromedarii ticks against oxidative damage. This finding will contribute to our understanding of the physiology of these ectoparasites and the development of untraditional methods to control them.     

Chemical identity of tryptensin with angiotensin.

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.     

The cellulase of Trichoderma viride: Separation of the components involved in the solubilization of cotton

1. Culture filtrates from Trichoderma viride have been fractionated by gel filtration on Sephadex G-75 followed by ion-exchange chromatography on DEAE- and SE-Sephadex. 2. The components essential for attack on cotton are a carboxymethylcellulase, a cellobiase and a third (C₁) component which has no action on CM-cellulose, cellobiose or cotton. 3. These components, which together can completely convert cotton into water-soluble products, lose this ability when separated and regain it quantitatively when recombined in their original proportions.     

Purification of an anticoagulant from the body fluid of Ascaris suum

An anticoagulant has been purified from the body fluid of Ascaris suum by sequential passage through Sephadex G-50, CM-cellulose and Sephadex G-25 columns then treated with 2 M NaCl, passaged through a Sephadex G-25 column, separated from the phosphate buffer by precipitation of the latter with the CaCl2, then passaged through a Sephadex G-10 column in water. In the body fluid of the worm, the anticoagulant is ionically-bound to a carrier substance. The complex can be split by treatment with 2 M NaCl. The molecular weight of the anticoagulant is slightly less than 1400.     

A simple and sensitive method of analysis for histamine,putrescine, and polyamines without the use of an amino acid analyzer

Histamine, putrescine, spermidine, and spermine in rat tissues and human urine were separated on a CM-cellulose column (0.6 × 10 cm). These amines in the chromatographic eluate were determined by the reactions with o-phthalaldehyde (for histamine), fluorescamine, o-phthalaldehyde-mercaptoethanol, or 2,4,6-trinitrobenzene sulfonate (for putrescine and polyamines). The procedures are rapid and simple when popular instruments are used. The limits of determination by the present method were of the order of 0.1 to 0.2 nmol for histamine and 2 to 4 nmol for putrescine and polyamines.     

An alkaline protease from fresh fruiting bodies of the edible mushroom <Emphasis Type="Italic">Pleurotus citrinopileatus</Emphasis>

A protease was purified from fresh fruiting bodies of the edible mushroom Pleurotus citrinopileatus. The isolation procedure included ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on CM-cellulose. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protease demonstrated a single band with a molecular mass of 28 kDa. The protease showed an optimal pH at 10 and an optimal temperature at 50°C. The activity of the protease was not affected by EDTA, indicating that it is not a metalloprotease. The protease exhibited a higher activity in the presence of K⁺ and Li⁺, but its activity was potently inhibited by Al³⁺, Cu²⁺, and Hg²⁺ ions. It manifested a K _m of 3.44 mg/ml and a V _max of 0.139 mg ml⁻¹ min⁻¹. It was devoid of ribonuclease and antifungal activities.     

Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures.

1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.     

Extracellular Lytic Enzymes of Myxococcus virescens II. Purification of Three Bacteriolytic Enzymes and Determination of their Molecular Weights and Isoelectric Points

The bacteriolytic enzymes produced by Myxococcus virescens and previously concentrated and separated from most of the non-bacteriolytic proteins have been further separated and purified. The bacteriolytic enzyme solution was concentrated by lyo-philization. When applied to a Sephadex G-100 column, three peaks of bacteriolytic activity were eluted. Polyacrylamide gel electrophoresis showed that all the three enzyme fractions were contaminated with at least four non-bacteriolytic proteins. In the first enzyme fraction the bacteriolytic enzymes could be freed from the contaminating proteolytic activity by adsorption on a hydroxylapatite column. The bacteriolytic enzymes could then be adsorbed on a CM-cellulose column. The remaining contaminating proteins passed the column un-adsorbed while the bacteriolytic enzymes could be eluted with a gradient of 0.02–0.10 M ammonium hydrogen carbonate solution. The second enzyme fraction was adsorbed on a CM-cellulose column and then eluted with 0.03–0.15 M NH4 HCO₃. After rechromatography on a new column under the same conditions, all of the contaminating proteins had disappeared. For purification of the third enzyme fraction chro-matography on one single CM-cellulose column was sufficient. The elution of the adsorbed enzymes was performed with a gradient of 0.15–0.30 M NH₄HCO₃. The recovery of activity for each of the ion-exchange chromatography separations was at least 90%. The purity of the enzymes was tested by polyacrylamid gel electrophoresis. Each of the purified enzymes gave only one coloured band which coincided with the enzyme activity assayed in sliced gels. The molecular weights of the enzymes were determined by electrophoresis on acryl-amide gels containing sodiumdodecylsulphate. The molecular weights determined in this way (about 40,000, 30,000 and 20,000, respectively) were about 10,000 daltons higher than those obtained by gel chromatography on Sephadex G-100. This discrepancy seems to depend on interactions between the enzymes and the dextran molecules probably caused by the strongly basic nature of the enzymes or by formation of enzyme-substrate complexes.     

N-acetylmuramyl-L-alanine amidase of Vi phage III.

1. A lytic enzyme was isolated from Vi phage III-induced lysate of Salmonella typhi, and purified about 200-fold by chromatography on IRC-50, CM-cellulose, and Sephadex G-75 columns. 2. Both E. coli B murein and muropeptide C6 were digested on incubation with the lytic enzyme. The main product of murein and muropeptide C6 digestion is identical with tetrapeptide Ala-Glu-DAP-Ala. The release of amino groups during digestion was not accompanied by the appearance of either reducing groups or hexosamines. 3. It is concluded that Vi phage III-induced lytic enzyme is N-acetylmuramyl-L-alanine amidase, which cleaves the amide bond between N-acetylmuramic acid and L-alanine.     

A diffusible factor responsible for the determination of cholinergic functions in cultured sympathetic neurons. Partial purification and characterization

A macromolecule has been partially purified which influences the choice of the neurotransmitter synthesized by sympathetic neurons. Previous studies showed that culture medium conditioned by incubation on certain types of non-neuronal cells increased [3H]acetylcholine synthesis and accumulation from [3H]choline in primary cultures of neurons dissociated from neonatal rat superior cervical ganglion and grown in the virtual absence of non-neuronal cells. A concomitant decrease of [3H]catecholamines production was observed (Patterson, P. H., and Chun, L. L. Y. (1977) Dev. Biol. 56, 263-280). The active cholinergic factor in conditioned medium from C6 glioma or primary rat heart cultures has been purified about 1500-fold by a sequence of ammonium sulfate precipitation, DEAE-, CM-cellulose, and Sephadex G-100 chromatography. The partially purified factor is active at 1 microgram of protein/ml of culture medium and is eluted from Sephadex columns as a single peak of apparent Mr = 40,000-45,000. This material is insensitive to 0.2 M 2-mercaptoethanol, but is inactivated by 1 mM Na periodate. Its activity is partially decreased by treatment with a protease from Streptomyces griseus but is unaffected by neuraminidase. Material purified through the ammonium sulfate, DEAE- and CM-cellulose steps contains large amounts of trypsin- and chymotrypsin-inhibiting activities.     

A0-phenylalanyl] relaxin (porcine): an active intermediate

A [phenylalanylA0] relaxin has been isolated as a byproduct during large scale porcine relaxin preparations, using ion exchange chromatography on CM-cellulose at pH 7.8 followed by high performance liquid chromatography on reversed phase columns. The elongation at the N terminus of the A-chain has been demonstrated by amino acid and sequence analyses of the isolated and carboxymethylated relaxin-A-chain. The phenylalanyl relaxin and B29 relaxin are indistinguishable by circular dichroism spectroscopy, in mouse pubic ligament assay, and radioimmunoassay. The occurrence of phenylalanyl relaxin may be caused by an incomplete conversion of prorelaxin to relaxin.     

Amino acid sequence of cardiotoxin-analogue I from the venom of Naja naja atra.

Cardiotoxin(CTX)-analogue I was obtained in a yield of about 5.3% from the venom of $\text{naja naja atra}$ by gel filtration on Sephadex G-50, followed by CM-cellulose column chromatography. When injected intraperitoneally in mice, its LD₅₀ was 2.8 (2.45–3.19) μg/g body weight. This toxin was cytotoxic to Yoshida sarcoma cells and produced contracture of the skeletal muscle as did CTX. The amino acid sequence of CTX-analogue I exhibits a high degree of homology with that of CTX from the same venom but differs in 13 positions.     

The characterization of the whey proteins of guinea-pig milk: The isolation and properties of α-lactalbumin

1. The whey proteins of guinea-pig milk were examined by electrophoresis on paper, cellulose acetate, starch gel and polyacrylamide gel. 2. Two major proteins were detected, one of which was identified as blood serum albumin. 3. The major whey protein was isolated by CM-cellulose chromatography and on columns of Sephadex G-100. 4. The amino acid composition of the protein, taken in conjunction with its other properties, indicated that the major whey protein in guinea-pig milk is homologous with cow α-lactalbumin and that β-lactoglobulin is absent from guinea-pig milk. 5. Guinea-pig α-lactalbumin, which was obtained crystalline, had mol.wt. 15800, N-terminal lysine and C-terminal glutamine.     

Studies on Bacterial Protease

A neutral protease of Bacillus subtilis var. amylosacchariticus was purified and crystallized by sequential chromatography on columns of Duolite A-2 anion-exchange resin, CM-cellulose and DEAE-sephadex A-50. The crystalline preparation was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria. The enzyme was most active at near pH 7 against casein and stabilized by calcium salts. Some metalchelating agents and metal ions such as Hg?, Pb?, Cu? and Fe? markedly inactivated the enzyme, whereas diisopropyl phosphorofluoridate, sulfhydryl reagents and protease inhibitor of potato did not affect the activity. The neutral protease obtained here was rather stable as compared with the neutral protease ever reported and was able to be freeze-dried without any appreciable lose in enzyme activity.