The molecular size of nematode acetylcholinesterases and their separation from nematode allergens

The molecular size of nematode acetylcholinesterases, and their separation from nematode allergens. International Journal for Parasitology 3: 735–741. Acetylcholinesterases and allergens were derived from two parasitic nematodes, Nippostrongylus brasiliensis which parasitises rats and Trichostrongylus colubriformis which infects sheep and guinea pigs.

Chromatographic studies indicated that the mol. wt of nematode acetylcholinesterases lies between 65,000 and 75,000. The acetylcholinesterases of both species were separated from nematode allergens whose mol. wt is in the range of 10,000–50,000.

γG binding antigens of T. colubriformis were located in fractions with a mol. wt range of 30,000–150,000. γE binding activity was confined to allergenic material with a mol. wt of less than 70,000.     

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

Limited proteolysis of glycogen phosphorylase a by subtilisin BPN'

The limited proteolysis of rabbit skeletal muscle phosphorylase a was undertaken with subtilisin BPN' immobilized to Sepharose 4B. The effect of substrates, activators and inhibitors of phosphorylase a was investigated by monitoring the changes in phosphorylase activity in the SDS gel electrophoretic pattern and in the 32P-content of 32P-labeled phosphorylase a. Phosphorylase a loses its activity upon subtilisin treatment. All ligands tested protect phosphorylase a activity against subtilisin action, probably by inducing structural changes in the tower loop of the enzyme. Glucose-6-P significantly accelerates [32P]peptide release from phosphorylase a through altering the structure of the N-terminal tail segment. The two subunits of dimeric phosphorylase a are held together by strong interactions--deduced from the correlation of the rate of proteolysis and the disappearance of catalytic activity.     

Features of glycogen phosphorylase from the body wall musculature of the lugworm Arenicola marina and the mode of activation during anoxia

The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 +/- 1.2 nmol . min-1 . (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 +/- 0.5 mumol . min-1 . (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5'-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

The stimulation of liver phosphorylase b by AMP, fluoride and sulfate. A technical note on the specific determination of the a and b forms of liver glycogen phosphorylase.

1. The activity of liver phosphorylase b from several mammalian species has been studied. The enzyme from rat or mouse has a higher activity than the rabbit enzyme, which is itself more active than pig liver phosphorylase b. 2 The activity of liver phosphorylase b is influenced by anions and by AMP, and these effects are influenced by pH. Fluoride, which is currently added to the assay mixture of phosphorylase a in crude preparations, is about as active as sulfate as a stimulator of phosphorylase b. 3. When assayed at pH 6.1 and in the presence of 0.15 M NaF, the activity of rat liver phosphorylase b reaches 25% of that of the a enzyme; if 1 mM AMP is also present, this value rises to 50%. 4. Methods are described that allow the determination of liver phosphorylase a without interference of b, and the determination of total phosphorylase (a+b) in rat liver.     

Phosphorylase kinase from chicken skeletal muscle. Quaternary structure, regulatory properties and partial proteolysis

Phosphorylase kinase has been purified from white and red chicken skeletal muscle to near homogeneity, as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular mass of the native enzyme, estimated by chromatography on Sepharose 4B, is similar to that of rabbit skeletal muscle phosphorylase kinase, i.e. 1320 kDa. The purified enzyme both from white and red muscles showed four subunits upon polyacrylamide gel electrophoresis in the presence of SDS, corresponding to alpha', beta, gamma' and delta with molecular masses of 140 kDa, 129 kDa, 44 kDa and 17 kDa respectively. Based on the molecular mass of 1320 kDa for the native enzyme and on the molar ratio of subunits as estimated from densitometric tracings of the polyacrylamide gels, a subunit formula (alpha' beta gamma' delta)4 has been proposed. The antiserum against the mixture of the alpha' and beta subunits of chicken phosphorylase kinase gave a single precipitin line with the chicken enzyme but did not cross-react with the rabbit skeletal muscle phosphorylase kinase. The pH 6.8/8.2 activity ratio of phosphorylase kinase from chicken skeletal muscle varied from 0.3 to 0.5 for different preparations of the enzyme. Chicken phosphorylase kinase could utilize rabbit phosphorylase b as a substrate with an apparent Km value of 0.02 mM at pH 8.2. The apparent V (18 mumol min-1 mg-1) and Km values for ATP at pH 8.2 (0.20 mM) were of the same order of magnitude as that of the purified rabbit phosphorylase kinase b. The activity of chicken phosphorylase kinase was largely dependent on Ca2+. The chicken enzyme was activated 2-4-fold by calmodulin and troponin C, with concentrations for half-maximal activation of 2 nM and 0.1 microM respectively. Phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (up to 2 mol 32P/mol alpha beta gamma delta monomer) and autophosphorylation (up to 8 mol 32P/mol alpha beta gamma delta monomer) increased the activity 1.5-fold and 2-fold respectively. Limited tryptic and chymotryptic hydrolysis of chicken phosphorylase kinase stimulated its activity 2-fold. Electrophoretic analysis of the products of proteolytic attack suggests some differences in the structure of the rabbit and chicken gamma subunits and some similarities in the structure of the rabbit red muscle and chicken alpha'.     

ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor

An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.     

Liver purine nucleoside phosphorylase in Camelus dromedarius: purification and properties

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyl transferase, EC 2.4.2.1) was purified to electrophoretic homogeneity from the liver of Camelus dromedarius. 2. The enzyme appears to be a dimer with a 44,000 subunit mol. wt and displays non-linear kinetics with concave downward curvature in double reciprocal plots with respect to both inosine and orthophosphate as variable substrates. 3. The effect of thiol compounds on the enzyme activity and of pH on kinetic parameters is reported.     

Preparation and properties of haemagglutinin from haemolymph of acrididae (Grasshoppers)

The proteinaceous haemagglutinin (lectin) present in the haemolymph of Melanoplus sanguinipes (F) has been isolated and biochemically characterized. The protein was purified to homogeneity by affinity chromatography on a column of Sepharose-galactose. The purified haemagglutinin accounted for all observed haemolymphatic haemagglutination activity. Gel filtration and electrophoresis showed the haemagglutinin to be a 600–700,000 mol. wt noncovalent aggregate of 70,000 mol. wt subunits. The 70,000 subunit contained two disulfide-linked polypeptide chains of mol. wt 40,000 and 28,000, respectively. The purified haemagglutinin contained all the common amino acids and had highest amounts of acidic residues and least amounts of methionine and glucosamine. Haemagglutination activity was stable at −20°C in the presence of d-galactose but was destroyed by treatment of the haemagglutinin with trypsin, heat or EDTA. Haemagglutination inhibition studies showed that low concentrations (≤5 mM) of either d-galactosidic or d-glucosidic carbohydrates were bound by the haemagglutinin and inhibited agglutination of human asialo-erythrocytes. The purified haemagglutinin was antigenic in rabbits. Comparative experiments showed that the haemagglutinin from M. differentialis (Thomas) was identical to that from M. sanguinipes.     

Influence of carbohydrate side chains on activity of tissue-type plasminogen activator

When messenger RNA (mRNA) from both untreated and phorbol ester-treated melanoma cells is translated in simple reticulocyte lysates, tissue-type plasminogen activator can be immunoprecipitated by an affinity-purified antibody as a approximately 52,000 mol wt protein, with no detectable biological (plasminogen activating) activity. When the reticulocyte lysate system is supplemented with a preparation of microsomal membranes, biological activity becomes detectable and a 63,000 mol wt protein can be immunoprecipitated with the same antibody. Furthermore, when natural tissue-type plasminogen activator (mol wt approximately equal to 70,000) is incubated with different glycosidases, distinct alterations in the electrophoretic mobility of the molecules are observed, together with alterations in the level of biological activity. While treatment with neuraminidase and beta-galactosidase caused decreases in activity, alpha-mannosidase caused an increase. These results suggest that the carbohydrate part of the molecule can influence its biological behavior.     

Purification and characterization of a glycogen phosphorylase analog missing the amino-terminal segment

A glycogen phosphorylase analog missing only the amino-terminal 16 to 18 residues, which include the phosphorylation site, was produced by subtilisin Carlsberg cleavage of phosphorylase b in the presence of caffeine. The analog, named phosphorylase b's, was purified, and its enzymatic properties were compared with those of phosphorylase b. The KM's for glucose 1-phosphate are similar, but phosphorylase b's has a VM 43% higher than that of phosphorylase b. Also, phosphorylase b's is less sensitive to inhibition by glucose 6-phosphate and stimulation by sodium fluoride than is phosphorylase b. The subunit interactions in the two enzyme forms were also compared. The monomer-monomer interactions in phosphorylase b's are weaker than in phosphorylase b, as evidenced by a faster rate of resolution of the coenzyme, pyridoxal phosphate, from phosphorylase b's. The dimer-dimer interactions are also weaker in phosphorylase b's than in phosphorylase b, because phosphorylase b's does not form tetramers or crystals as readily as does phosphorylase b. Because removal of the amino-terminal segment changes the properties of the enzyme, this segment must be interacting with other parts of the protein. This statement conflicts with previous interpretation of X-ray crystallographic data that suggest that the amino-terminal region of phosphorylase b is freely mobile. Possible explanations for this contradiction are discussed.     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons

The possibility that proteins are modified during axoplasmic transport in central nervous system axons was examined by analyzing neurofilament proteins (200,000, 140,000, and 70,000 mol wt) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFPs) exhibited considerable microheterogeneity. At least three forms of the “ 140,000” neurofilament protein differing in molecular weight by SDS PAGE (140,000-145,000 mol wt) were identified. The “140,000” proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The “140,000” NFPs exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 mol wt) were present along the entire length of the optic pathway. The third protein (143,000 mol wt) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [(3)H]proline, newly synthesized radiolabeled proteins in the “140,000” mol wt region entered proximal mouse retinal ganglion cell (RGC) axons as two major species corresponding to the 145,000 and 14,000 mol wt NFPs observed on stained gels. When transported NFPs reached more distal axonal regions (30 d postinjection or longer), a 143,000 mol wt protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 mol wt species. The results suggest that (a) the composition of CNS neurofilaments, particularly the “140,000” component, is more complex than previously recognized, that (b) retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins, and that (c) structural heterogeneity of “140,000” NFPs arises, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a calcium-dependent enzymatic process active at endogenous calcium levels. Changes in major proteins other than those in the 145,000-140,000 mol wt region were minimal. In optic pathways from mice injected intravitreally with L-[(3)H]proline, tritiated 143,000 mol wt NFP formed rapidly in vitro if radioactively labeled NFPs were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 mol wt NFP was generated if radioactively labeled NFPs were present proximally in RGC axons (6 d postinjection). The enzymatic process that generates 143,000 mol wt NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. The foregoing results and other observations, including the accompanying report (J. Cell Biol., 1982, 94:159-164), imply that CNS axons may be regionally specialized with respect to structure and function.     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

cAMP-dependence of phosphorylation of the phosphorylase b kinase of the skeletal muscles of rats during physical exercise and training

During relative rest of trained rats the phosphorylase b kinase activity is increased by 24% (pH 6.8). Physical load causes an increase of the phosphorylase b kinase activity of untrained and trained rats by 44 and 33%, respectively. The degree of phosphorylase b kinase phosphorylation by a homologous soluble cAMP-dependent protein kinase from the muscles of trained rats at rest is 1.9 times that of the control group. The cAMP-dependent phosphorylation of phosphorylase b kinase of untrained and trained rats under physical load is increased 2.5-fold. The data obtained are indicative of the regulatory role of cAMP-dependent phosphorylation in biochemical adaptation of skeletal muscles when their function is increased.     

Phosphorylase phosphatase catalytic subunit. Evidence that the Mr = 33,000 enzyme fragment is derived from a native protein of Mr = 70,000

An active form of phosphorylase phosphatase of Mr = 33,000, referred to as the catalytic subunit for over a decade, was purified to near-homogeneity from rabbit skeletal muscle. Repeated immunization of a sheep produced immunoglobulins that blocked the activity of the phosphatase. These immunoglobulins were affinity-purified on columns of immobilized phosphorylase phosphatase and used as macromolecular probes in a "Western" immunoblotting procedure with peroxidase-conjugated rabbit anti-sheep immunoglobulins. Only one protein, of Mr = 33,000, was stained in samples of the immunogen, attesting to the specificity of the probes. However, the Mr = 33,000 phosphatase protein was not detected in muscle extracts or in partially purified preparations. Instead, a single protein of Mr = 70,000 was detected. Limited proteolysis, in particular by Staphylococcus aureus V8 protease and thermolysin, converted the immunoreactive protein from Mr = 70,000 to Mr = 33,000. Coagulation of the phosphatase preparation with 80% ethanol at room temperature rendered the Mr = 70,000 protein insoluble, but allowed extraction of the Mr = 33,000 protein from the precipitate. Thus, we conclude that the immunoreactive protein of Mr = 70,000 is the "catalytic subunit" of phosphorylase phosphatase with a catalytic domain of Mr = 33,000. Previous purification schemes have yielded only the fragment of Mr = 33,000 due to its relative resistance to proteolysis and coagulation. Gel filtration chromatography of the "native" form of phosphorylase phosphatase showed Mr approximately 230,000. Both the Mr = 70,000 catalytic subunit and a Mr = 60,000 protein related to inhibitor-2 were detected by immunoblotting in the same fractions that exhibited activity after treatment with Co2+ and trypsin. Only the Mr = 60,000 protein was degraded during this activation process. We propose that the native phosphorylase phosphatase is an elongated structure with two-fold symmetry, containing one catalytic subunit of Mr = 70,000 and one regulatory subunit of Mr = 60,000.     

Antifungal Hydrolases in Pea Tissue : I. Purification and Characterization of Two Chitinases and Two beta-1,3-Glucanases Differentially Regulated during Development and in Response to Fungal Infection

Chitinase and β-1,-3-glucanase activities increased coordinately in pea (Pisum sativum L. cv “Dot”) pods during development and maturation and when immature pea pods were inoculated with compatible or incompatible strains of Fusarium solani or wounded or treated with chitosan or ethylene. Up to five major soluble, basic proteins accumulated in stressed immature pods and in maturing untreated pods. After separation of these proteins by chromatofocusing, an enzymic function could be assigned to four of them: two were chitinases and two were β-1,3-glucanases. The different molecular forms of chitinase and β-1,3-glucanase were differentially regulated. Chitinase Ch1 (mol wt 33,100) and β-1,3-glucanase G2 (mol wt 34,300) were strongly induced in immature tissue in response to the various stresses, while chitinase Ch2 (mol wt 36,200) and β-1,3-glucanase G1 (mol wt 33,500) accumulated during the course of maturation. With a simple, three-step procedure, both chitinases and both β-1,3-glucanases were purified to homogeneity from the same extract. The two chitinases were endochitinases. They differed in their pH optimum, in specific activity, in the pattern of products formed from [³H]chitin, as well as in their relative lysozyme activity. Similarly, the two β-1,3-glucanases were endoglucanases that showed differences in their pH optimum, specific activity, and pattern of products released from laminarin.     

The subunit structure of Daphnia pulex hemoglobin

• 1.1. The extracellular hemoglobin of Daphnia pulex has an apparent molecular weight of 430,000–470,000 by gel chromatography and an S_20,w = 16.9 at pH 7.0.
• 2.2. Purified hemoglobin contains one heme per 18,000–20,000 g protein. The polypeptide chains are heterogeneous with mol. wts between 31,000–37,000. Some high mol. wt (M_r = 53,000–86,000) material is also present.
• 3.3. The hemoglobin dissociates at pH 10.5 in EDTA into 3S material which can be digested with subtilisin into 16,000 mol wt heme-containing polypeptide chains.
• 4.4. The amino acid composition of the intact hemoglobin is identical to that of the heme-containing fragments generated by proteolytic digestion of the 3S material.
• 5.5. These results are consistent with the hypothesis that D. pulex hemoglobin is composed of subunits containig two heme groups per 35,000 mol. wt polypeptide chain.

     

Physicochemical and immunochemical studies on bovine IgA and glycoprotein-a

1. Bovine secretory IgA (SIgA) from colostrum (mol. wt. about 410,000) is composed of four alpha-chains (mol. wt. 61,000), four light chains (mol. wt. 23,000) and one molecule of glycoprotein-a (mol. wt. 70,000-86,000). The alpha-chains are antigenically and physicochemically distinct from the heavy chains of IgM, IgG1 and IgG2 while the light chains are identical to those occurring on other bovine immunoglobulins. Glycoprotein-a and bovine free secretory component are identical and the former name should be abolished. Much of this protein is covalently bonded to IgA. 2. The gel filtration behavior of serum IgA suggests it is a dimer. 3. The elution behavior of IgA and SIgA from ion-exchange columns and the solubility characteristic of SIgA in the presence of Zn2+ are similar to those of human and rabbit IgA. 4. The disc electrophoretic behavior of IgA and SIgA are distinct from IgM, dimeric IgG1, 7-S IgG and glycoprotein-a. Dimeric IgG1 (s20,w = 9.5) is abundant in colostrum and is similar in size to SIgA. 5. Bovine IgA shows physicochemical and immunochemical heterogeneity when studied by gel filtration, disc electrophoresis, immunoelectrophoresis and ultracentrifugational analyses. Lacrimal and nasal SIgA possess antigenic determinants absent on colostral SIgA.     

Interaction of flavonoids with rabbit muscle phosphorylase kinase

We have examined the effect of several flavonoids on the activity of phosphorylase kinase from rabbit skeletal muscle. From 14 flavonoids tested, the flavones quercetin and fisetin were found to be efficient inhibitors of nonactivated phosphorylase kinase when assayed at pH 8.2, causing 50% inhibition at a concentration of about 50 microM, while the flavanone hesperetin stimulated phosphorylase kinase activity about 2-fold when tested at 250 microM. The efficiency of quercetin in inhibiting the kinase is higher when the enzyme is stimulated either by ethanol or by alkaline pH. Both casein and troponin phosphorylation by phosphorylase kinase and the autophosphorylation of the kinase were inhibited by quercetin. In addition, quercetin was found to be a competitive inhibitor of ATP for the phosphorylation of phosphorylase b at pH 8.2. These observations suggest that the inhibitory effect of the flavone is directly on the phosphorylase kinase molecule. Trypsin-activated phosphorylase kinase was inhibited by quercetin and stimulated by hesperetin, as for the native enzyme.