Carp myogens of white and red muscles. Gross isolation on sephadex columns of the low-molecular-weight components and examination of their participation in anaerobic glycogenolysis

1. The combined low-molecular-weight protein components of the myogens from carp white and red muscles [about 30% (w/w) of the myogen proteins] have been isolated by gel filtration on Sephadex G-75 columns. 2. The presence in this fraction from myogen of white muscle of the three main electrophoretic components previously isolated has been confirmed, and the low molecular weight of the fourth component has been definitely established. 3. The exclusive presence of this fourth component in the myogen of red muscle, apart from myoglobin, has also been demonstrated. 4. Glycogenolysis experiments in vitro have shown that the low-molecular-weight protein fraction from carp myogen does not contain enzymes from the Embden–Meyerhof chain.     

Carp myogens of white and red muscles. Properties and amino acid composition of the main low-molecular-weight components of white muscle

1. The three main components of the 1·5–2s ultracentrifugal peak of carp myogen (white muscle) have been isolated by ammonium sulphate fractionation and zone electrophoresis, and crystallized. 2. The molecular weights of these three proteins were determined by sedimentation and diffusion, by the Archibald method and by amino acid analysis, and found to lie between 9000 and 13000. 3. Their complete amino acid compositions were determined by column chromatography and by their ultraviolet spectra. Both methods revealed abnormal compositions, including the absence of tryptophan and methionine and the presence of large amounts of phenylalanine. At most 1 residue each of tyrosine, cysteine, proline, arginine and histidine was found/molecule. 4. The specific viscosity of component 3 was lower than that of other small globular proteins described so far, a fact that suggests that these proteins approximate more closely to the ideal case of the spherical protein molecule. Also, the presence of a single residue of several amino acids, the absence of disulphide bonds, and the apparent reversibility of denaturation by urea of component 3 suggest that the study of these molecules could provide new information on the structure of proteins.     

Separation and properties of β-galactosidase, β-glucosidase, β-glucuronidase and n-acetyl-β-glucosaminidase from rat kidney

1. The activities of β-galactosidase, β-glucosidase, β-glucuronidase and N-acetyl-β-glucosaminidase from rat kidney have been compared when 4-methylumbelliferyl glycosides are used as substrates. 2. Separation by gel electrophoresis at pH7·0 indicated slow- and fast-moving components of rat-kidney β-galactosidase. 3. The fast-moving component is also associated with the total β-glucosidase activity and inhibition experiments indicate that a single enzyme species is responsible for both activities. 4. DEAE-cellulose chromatography and filtration on Sephadex gels suggests that the β-glucosidase component is a small acidic molecule, of molecular weight approx. 40000–50000, with optimum pH5·5–6·0 for β-galactosidase and β-glucosidase activities. 5. The major β-galactosidase component has low electrophoretic mobility, a calculated molecular weight of 80000 and optimum pH3·7.     

Interactions between glycosaminoglycans and proteins, with particular reference to a new technique for isolating serum glycoproteins

1. Chondroitin sulphate–serum protein complexes (A, B and C), successively precipitated by adding chondroitin sulphate to serum at three arbitrary descending pH values (5·2, 4·3 and 3·1), were dissociated at pH 6·7 and chromatographed on DEAE-Sephadex, when the liberated serum proteins were simultaneously freed of chondroitin sulphate and separated into five fractions. Evidence that serum proteins were precipitated as a result of electrostatic interactions with dissociated carboxylate groups on the glycosaminoglycan is presented. 2. Serum proteins (fraction G), unable to form complexes with chondroitin sulphate, contained 4·4% of sialic acid and accounted for 4 and 26% of the total protein and protein-bound sialic acid in serum respectively. This fraction interacted electrostatically with chondroitin sulphate only when rendered more basic by removal of sialic acid residues with neuraminidase. The heat stability, solubility properties and high carbohydrate content of fraction G classified it as a seromucoid fraction. 3. Fraction G contained several glycoprotein and hexuronic acid-containing fractions, including a hitherto undetected brown-pigmented high-molecular-weight serum component, which migrated in starch gel between the origin and Sα₂-globulin and contained 3·1 and 4·1% of sialic acid and hexuronic acid respectively. 4. Glycosaminoglycan–protein interactions are discussed in relation to protein fractionation. By prior removal of less acidic proteins by these interactions, a new technique is available for isolating serum seromucoids in higher yields and under milder conditions than existing methods.     

THE LYSOSOME PERIPHERY: BIOCHEMICAL AND ELECTROKINETIC PROPERTIES OF THE TRITOSOME SURFACE

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm², and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10^-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 µg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 ± 3; glucosamine, 24 ± 3; galactosamine, 10 ± 2; glucose, 21 ± 2; galactose, 26 ± 2; mannose, 31 ± 5; fucose, 7 ± 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.     

Purification and properties of diaminopimelate decarboxylase from Escherichia coli

1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu²⁺, Hg²⁺) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate.     

Stimulation of adenine phosphoribosyltransferase by adenosine triphosphate and other nucleoside triphosphates

1. Adenine phosphoribosyltransferase was protected from inactivation on heating at 55° by the presence of 5-phosphoribosyl pyrophosphate. ATP, adenine, AMP or GMP had no protective effect on the activity of this enzyme. The presence of either 5-phosphoribosyl pyrophosphate or ATP did not protect adenine phosphoribosyltransferase against the loss of ATP stimulation obtained by heating at 55°. 2. At pH5·3 and 6·0 adenine phosphoribosyltransferase was stimulated by a narrow range of ATP concentration (15–25μm). At pH6·5 and 7·0 maximum stimulation was obtained with 25–30μm-ATP, and at pH7·4, 8·2 and 8·85 maximum stimulation was obtained over a wide range of ATP concentrations (60–200μm). With extracts that had been heated for 30min. at 55° no stimulation was observed at either pH5·3 or 7·4 with ATP concentrations up to 100μm. 3. Short periods of heating at 55° (1, 2 or 5min.) increased the stimulation of adenine phosphoribosyltransferase obtained with various concentrations of ATP. 4. The addition of CTP, GTP, deoxy-GTP, deoxy-TTP or XTP to assay mixtures resulted in weak stimulation of adenine-phosphoribosyltransferase activity. 5. It is suggested that there are at least three different forms of adenine phosphoribosyltransferase, each with a different affinity for ATP.     

Kinetics of thiamin cleavage by sulphite

Results are presented on the rate of thiamin cleavage by sulphite in aqueous solutions as affected by temperature (20–70°), pH(2·5–7·0), and variation of the concentration of either thiamin (1–20μm) or sulphite (10–5000μm as sulphur dioxide). Plots of the logarithm of percentage of residual thiamin against time were found to be linear and cleavage thus was first-order with respect to thiamin. At pH5 the rate was also found to be proportional to the sulphite concentration. In the pH region 2·5–7·0 at 25° the rate constant was 50m⁻¹hr.⁻¹ at pH5·5–6·0, and decreased at higher or lower pH values. The rate of reaction increased between 20° and 70°, indicating a heat of activation of 13·6kcal./mole.     

Hydrolysis of fibrous cotton and reprecipitated cellulose by cellulolytic enzymes from soil micro-organisms

1. The catalytic decomposition of undegraded cellulose in the form of cotton fibres is described with hydrogen peroxide at 0·4–0·04% (w/v) concentration in the presence of ferrous salts at pH3–5. 2. Complete solubilization of 5mg. of cotton fibres occurred in about 7 days in the presence of 0·4% hydrogen peroxide and 0·2mm-ferrous sulphate at the optimum pH4·2–4·3. 3. With 0·4% hydrogen peroxide the most rapid decomposition of cellulose was confined to ferrous sulphate concentrations of approx. 2–0·02mm. If the concentrations of the reagents were decreased in proportion extensive breakdown occurred but much more slowly. 4. In the primary stages of breakdown cotton fibres were disintegrated to very short fibres. These were subsequently solubilized, but there was little accumulation of soluble material. Organic matter was lost from solution as the reaction progressed. 5. Other naturally occurring cellulose-containing materials, such as grass, straw, hay and sawdust, were also disintegrated and solubilized by hydrogen peroxide and ferrous sulphate.     

Rat-kidney lysosomes: isolation and properties

1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of α-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate α-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal α-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6·2. 4. The stability of lysosomal suspensions was studied. Incubation at 37° and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the α-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6–7 and less stable at pH 8–9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0·6m to 0·25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.     

Purification of rabbit kidney cytokinase and a comparison of its properties with human urokinase

1. The cytokinase (tissue activator of plasminogen) content of several mammalian tissues was evaluated by a quantitative casein hydrolysis method. 2. An alkaline (pH10·5) extraction of cytokinase from rabbit kidney lysosome–microsome fraction, followed by chromatography on DEAE-cellulose at pH7·6 with stepwise or linear increase in concentration of phosphate buffer, gave an 86-fold purification of the enzyme. The purified material was non-proteolytic against casein and heated fibrin and was freeze-dried without significant loss of activity or solubility. 3. Cytokinase is a protein with E^0·1%_1cm.=0·87 at 280mμ, and does not possess sufficient hexose or sialic acid to be classified as a glycoprotein. It has S_20,w 2·9–3·1s and molecular weight 50000 when measured on a calibrated Sephadex G-100 column. It has an isoelectric point between pH8 and pH9, and is maximally active and stable at pH8·5. It is inactivated by heat at 78°. 4. Cytokinase and human urokinase have the same K_m value and are inhibited in a partially competitive manner by -aminohexanoic acid and aminomethylcyclohexanecarboxylic acid. They are also inhibited by cysteine and arginine, but are unaffected by iodoacetamide and p-chloromercuribenzoate. 5. On the basis of this and other evidence it is suggested that rabbit kidney cytokinase and human urokinase are similar, if not identical, enzymes.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

Catalytic decomposition of cellulose under biological conditions

1. The catalytic decomposition of undegraded cellulose in the form of cotton fibres is described with hydrogen peroxide at 0·4–0·04% (w/v) concentration in the presence of ferrous salts at pH3–5. 2. Complete solubilization of 5mg. of cotton fibres occurred in about 7 days in the presence of 0·4% hydrogen peroxide and 0·2mm-ferrous sulphate at the optimum pH4·2–4·3. 3. With 0·4% hydrogen peroxide the most rapid decomposition of cellulose was confined to ferrous sulphate concentrations of approx. 2–0·02mm. If the concentrations of the reagents were decreased in proportion extensive breakdown occurred but much more slowly. 4. In the primary stages of breakdown cotton fibres were disintegrated to very short fibres. These were subsequently solubilized, but there was little accumulation of soluble material. Organic matter was lost from solution as the reaction progressed. 5. Other naturally occurring cellulose-containing materials, such as grass, straw, hay and sawdust, were also disintegrated and solubilized by hydrogen peroxide and ferrous sulphate.     

Partial characterization of the sialic acid-free forms of α1-acid glycoprotein from human plasma

Some of the properties of sialic acid-free α₁-acid glycoprotein prepared by mild acid hydrolysis (pH1·6 at 80° for 1hr.) were compared with those of neuraminidasetreated α₁-acid glycoprotein. Chemically, the former contained less fucose (15%) and amide (2%) residues. Physicochemically, it had undergone certain changes primarily pertaining to the secondary structure, so that the specific optical rotation was more negative than that of the latter. A further expression of this change is probably the difference in the pH range of the resolution into two bands on electrophoresis. The resolution of the glycoprotein prepared by mild acid hydrolysis seems to be extended to more acidic pH values both by starch-gel and free moving-boundary electrophoresis. On ultracentrifugation both preparations appeared homogeneous and sedimented with a rate of 3s. Removal of sialyl residues at different pH values, in the range 1–7, showed that 2moles of sialic acid/mole of protein are very strongly bound. The two variants of α₁-acid glycoprotein were isolated from pooled sialic acid-free α₁-acid glycoprotein by preparative starch-gel electrophoresis and from selected blood of normal adults by fractionation by solubility and chromatography. Ultracentrifugal and starch-gel electrophoretic analyses at pH5, with incubation times of 1 or 24hr., demonstrated that no dissociation–association equilibrium (constant sedimentation coefficient and molecular weight) or isomerization (constant apparent electrophoretic mobilities) exist between the two variants. Therefore these variants are not sub-units of native α₁-acid glycoprotein but represent modifications of naturally occurring proteins. Further, it was shown that the difference in the electrophoretic mobilities between the two variants was not due to any difference in amide content. Immunochemically, the two variants share the same determinants.     

The isolation and characterization of subcellular components of the epithelial cells of rabbit small intestine

1. Homogenization of the epithelial cells of rabbit small intestine in 0·3m-sucrose–5mm-EDTA, pH7·4, maintains intact the microvillus sheets that form the lumenal surface of the cells, the nuclei, the mitochondria and the vesicles (microsomes) formed from the endoplasmic reticulum. 2. These particulate components of the cell, and the cell-sap fraction, have been isolated by differential centrifuging of cell homogenates. 3. The nuclei and microvillus sheets sediment together and it has been impossible to separate these subcellular components by centrifugal methods. 4. The isolated subcellular fractions have been identified by a combination of light-microscopic examination, electron-microscopic examination, chemical analysis and assay for selected enzyme activities.     

Electrophoretic properties of ovomucoid

1. The nature of the electrophoretic heterogeneity of ovomucoid was investigated. Optimum resolution of the fractions on starch-gel electrophoresis occurred over a narrow range of pH and ionic strength. The pattern was not altered in the presence of 8m-urea but the bands were sharper. Ovomucoid–trypsin complex is stable at pH4·6 but dissociated in 6m-urea. 2. The two major fractions of ovomucoid were eluted from the gels. One of these was virtually free of sialic acid and the other, which contained 0·4mole of sialic acid/mole of protein, split into two components on electrophoresis after neuraminidase treatment. It was concluded that these two components, and likewise the two major fractions of ovomucoid, differ by a unit charge/mol. Differences in sialic acid content account for only part of the electrophoretic heterogeneity of ovomucoid.     

Solution properties of polygalacturonic acid

1. The specimen of polygalacturonic acid used in these studies was shown to contain very little neutral sugar, methyl ester groups or ash, and only residues of galacturonic acid. Its electrophoretic homogeneity was examined in pyridine–acetic acid buffer at pH6·5 and in borate buffer at pH9·2. The distribution of effective particle weights was shown to be fairly narrow. 2. The pH-titration curve of the polymer gave a pK value of 3·7. 3. The interaction of the polymer with Ruthenium Red was studied and titration curves were obtained for the spectral shifts associated with the formation of a complex. 4. Optical-rotatory-dispersion studies showed that the Drude constant, λ_c, was dependent on pH. 5. Polygalacturonic acid was shown to display non-Newtonian properties in solution and to have an anomalously high relative specific viscosity at low concentrations. 6. Studies were made of the pH-dependence of the sedimentation coefficient of the polymer. 7. These results are discussed in terms of the structure of the molecule and their relevance to the properties of pectic substances.     

The action of semicarbazide on the aggregation of the tropocollagen macromolecule

1. A difference in conformation was found between the collagen in solutions treated with semicarbazide hydrochloride and those treated with sodium chloride. This difference could be correlated with the difference in extent of aggregation between the fibrils precipitated from these solutions. 2. The action of semicarbazide hydrochloride depended on the pH and temperature of treatment in a complex manner. At constant temperature semicarbazide enhanced aggregation at pH values less than 4·3, but decreased aggregation was observed at pH values greater than 5·0. At pH 4·3 the effect of semicarbazide on aggregation varied with temperature, the tendency to increased aggregation being more pronounced at 34° and 36–37°. Similar increased aggregation tendencies superimposed on an overall decreased aggregation were observed at these temperatures at pH8·9. 3. A specific binding of semicarbazide to the collagen molecule was indicated.     

Enzymes and cancer: Preparation and some properties of guanase from rabbit liver

1. Guanase has been purified 200-fold in 20% yield from the supernatant fraction of rabbit-liver homogenates, by using ammonium sulphate fractionation, calcium phosphate-gel adsorption and chromatography on DEAE-cellulose and Sephadex G-200. 2. K_m with guanine as substrate at the optimum pH of 7·7 was found to be 1·05×10⁻⁵m. Q₁₀ was 1·4 between 23° and 48°. 3. Substrate activity and pH optima of compounds related to guanine have been studied. 8-Azaguanine, 1-methylguanine, thioguanine and 1-methylthioguanine are all substrates.     

Partition of porphyrins between cyclohexanone and aqueous sodium acetate as a function of pH. Determination of uroporphyrin and of hydrophilic porphyrin conjugates

1. The partition of uroporphyrins I and III, coproporphyrins I and III, haematoporphyrin IX, porphyrin c and a hydrophilic porphyrin–peptide fraction from variegate-porphyria faeces has been studied in systems of equal volumes of cyclohexanone and sodium acetate buffers of varying pH and concentration. 2. The concentration of acetate in the aqueous phase has little effect on the partition of porphyrin c, but markedly influences that of uroporphyrin. At 50% acetate saturation and pH4·5, only 5% enters the cyclohexanone phase whereas 60% of porphyrin c is extracted under similar conditions. 3. This circumstance forms the basis of a method for the determination of hydrophilic porphyrin–peptides in variegate-porphyria urine. Its reliability has been checked in model experiments. 4. At pH1·5 and an aqueous phase half-saturated with sodium acetate, an equal volume of cyclohexanone removes 95–97% of uroporphyrin and about 55% of porphyrin c. Uroporphyrin may therefore be determined as a second step in the method. 5. For the routine determination of uroporphyrin in systems free from other hydrophilic porphyrins, cyclohexanone extraction may be performed at any pH in the range 1·0–3·0.