Evidence for dissociation of ferrihemoglobin by poly-L-lysine

Summary Circular dichroism and absorption spectra of ferrihemoglobin were shown to be altered upon binding with poly-L-lysine at alkaline pH. When ferrihemoglobin immobilized to Sepharose gel was treated with poly-L-lysine, hemoglobin subunits were released from the gel. These results suggest that ferrihemoglobin was dissociated into subunits by poly-L-lysine.We thank Dr A. Ichihara and K. Aki for their valuable discussions and encouragement during the course of these studies.     

Changes in rate of methemoglobin reduction and oxygen affinity of erythrocytes incubated with inosine,pyruvate and phosphate

Summary Incubation of human erythrocytes with inosine, pyruvate and phosphate increases several fold the ferrihemoglobin reductase activity, the values of which, however, depend on the age of blood (by 6 to 2 times with respect to the normal value of fresh blood).     

Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with 125iodine.

In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with 125Iodine:chloramine T, lactoperoxidase, and an original technique of 'self labeling' based on the ability of the enzyme to oxidize and bind 125I in the presence of H2O2. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 microCi/micrograms MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (less than or equal to 3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits.     

Molecular weight of human alpha2-H-ferroglobulin subunits. Comparison with molecular weight of ferritin subunits]

alpha2 H globulin, a glycoferroprotein, was first demonstrated in the sera of patients with malignant diseases. This protein was isolated from cancerous human liver, and compared with ferritin, a ferroprotein showing some identical properties (presence of iron, high molecular weight, common antigenic determinants). However, physicochemical differences were observed between these two proteins. The study of protein dissociation was performed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after reduction by mercaptoethanol. A similar molecular weight of 19 000 is obtained for subunits of these two proteins. This value agrees well with the results obtained by other authors for ferritin.     

Polycationic modified polypeptides enhancing poly I:C induced viral resistance.

Polycationic modified derivatives of polyglutamic acid are at least as good enhancers of poly I:C induced viral resistance in various cell cultures as are DEAE-dextran or poly-L-lysine.     

Purification and characterization of aβ-glucosidase (linamarase) from the haemolymph ofZygaena trifolii Esper, 1783 (Insecta,Lepidoptera)

Summary A -glucosidase (linamarase) was purified 52-fold with a recovery of 27% from the haemolymph of the larvae ofZygaena trifolii, ESPER, 1783 (Lepidoptera, Zygaenidae). The final enzyme preparation was found to be nearly homogeneous on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be about 130 kDa; it consisted of two subunits of about 66 kDa. The enzyme showed an optimum between pH 4.5 and 5 with linamarin and a broad optimum between pH 3.5 and 6.5 for p-nitrophenyl--D-glucoside; the temperature optimum was 40°C. The -glucosidase showed a high specificity for its endogenous substrates linamarin and lotaustralin. Among the other natural and artificial substrates tested, only prunasin and p-nitrophenyl--D-glucoside were hydrolyzed by the enzyme, whereas linustatin, salicin, cellobiose and trehalose were not. The enzyme is strongly inhibited by -glucosylpiperidine.     

Self-labeling of human polymorphonuclear leucocyte myeloperoxidase with125iodine

In order to obtain a radioimmunoassay (RIA) technique for the measurement of human plasma myeloperoxidase (MPO), we purified the enzyme from polymorphonuclear granulocytes (neutrophils), and compared three methods of labeling it with¹²⁵Iodine: chloramine T, lactoperoxidase, and an original technique of self labeling based on the ability of the enzyme to oxidize and bind¹²⁵I in the presence of H₂O₂. The chloramine T technique produced a degraded protein, as well shown by a high non-specific binding of tracer to antibody. The lactoperoxidase technique did not succeed in labeling MPO with an adequate specific activity. In contrast, the self-labeling method gave a stable tracer with a specific activity of 23 Ci/gmg MPO (85 MBq), a satisfactory level of immunoreactivity, and a low-specific binding (3%). After labeling, purification of tracer was achieved by gel filtration chromatography in phosphate buffer (0.05 M; pH7) to which 0.1% poly-L-lysine was added. The labeled molecule remained stable for 40 days and could be used for RIA with a polyclonal antibody raised in rabbits.     

大鼠侧脑室下区神经干细胞具有不易兴奋性

目的建立体外培养大鼠侧脑室下区神经干细胞的方法,观察大鼠侧脑室下区神经干细胞的膜兴奋性。方法无血清培养方法体外分离、纯化孕15～16dwistar胎鼠的侧脑室下区神经干细胞,用免疫荧光鉴定干细胞标记蛋白nestin表达情况、用tuj-1和GFAP免疫染色研究体外NSC的分化情况;取第二代神经干细胞给予DiBACA（3）染色后,经高浓度氯化钾刺激,激光共聚焦显微镜动态扫描,观察侧脑室神经干细胞的兴奋性。结果采用无血清培养基体外分离的神经干细胞具有自我增殖、多向分化潜能等干细胞一般特点,且表达干细胞的标记蛋白nestin;采用DiBAC4（3）染色,高浓度钾刺激后,细胞荧光强度无显著变化,即细胞膜电位无明显改变,神经干细胞具有不易兴奋性。结论采用无血清培养方法成功分离扩增大鼠脑内神经干细胞;由大鼠侧脑室分离而来的神经干细胞具有不易兴奋性。     

Protein kinase CK2 and new binding partners during spermatogenesis

Protein kinase CK2 is an ubiquitously expressed enzyme that is absolutely necessary for the survival of cells. Besides the holoenzyme consisting of the regulatory β-subunit and the catalytic α- or α′-subunit, the subunits exist in separate forms. The subunits bind to a number of other cellular proteins. We show the expression of individual subunits as well as interaction with the transitional nuclear protein TNP1 and with the motor neuron protein KIF5C during spermatogenesis. TNP1 is a newly identified binding partner of the α-subunit of CK2. CK2α and KIF5C were found in late spermatogenesis, whereas CK2β and TNP1 were found in early spermatogenesis. CK2α, CK2α′, TNP1, and KIF5C were detected in the acrosome of spermatozoa, while CK2β was detectable in the mid-piece. Combinations of CK2 subunits might determine interactions with other proteins during spermatogenesis. KIF5C as a kinesin motor neuron protein is probably involved in the redistribution of proteins during spermatogenesis.     

Preparations of human dopamine beta hydroxylase subunits and antibodies against these subunits (author's transl)]

The present study deals with the dissociation of human dopamine beta hydroxylase (DBH) in subunits with a mol. wt of 79,500, and the preparation of specific antibodies in rabbits. No cross-reactivity was observed between human DBH and antibodies against human DBH subunits.     

Vacuolar H+-ATPase: From mammals to yeast and back

Vacuolar H⁺-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V₁) and membrane (V₀) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPases that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by drinking the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.     

A hydrogen-bonding network in mammalian sorbitol dehydrogenase stabilizes the tetrameric state and is essential for the catalytic power

Subunit interaction in sorbitol dehydrogenase (SDH) has been studied with in vitro and in silico methods identifying a vital hydrogen-bonding network, which is strictly conserved among mammalian SDH proteins. Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. The results focus on a network of coupled hydrogen bonds in wild-type SDH that uphold the protein interface, which is specific and favorable to electrostatic, van der Waals and hydrogen-bond interactions between subunits, interactions that are crucial for the catalytic power of SDH. Received 13 July 2007; received after revision 30 September 2007; accepted 1 October 2007     

Purification of canine myocardial mitochondrial creatine kinase

Summary Mitochondrial creatine kinase, purified for the first time, is a dimeric molecule with a mol.wt of 82,000 and does not hybridize with M or B subunits or react to their specific antiserum. A specific antiserum to the mitochondrial form was developed which does not cross-react with the B or M subunits. Thus, the mitochondrial form is biochemically and immunologically unique.Supported in part by the Special Center of Research in Ischemic Heart Disease (1 P17 HL 17646).I thank Ann Grace for her technical assistance and Ava Ysaguirre for typing the mansucript.     

G protein βγ subunits: Central mediators of G protein-coupled receptor signaling

G protein betagamma subunits are central participants in G protein-coupled receptor signaling pathways. They interact with receptors, G protein alpha subunits and downstream targets to coordinate multiple, different GPCR functions. Much is known about the biology of Gbetagamma subunits but mysteries remain. Here, we will review what is known about general aspects of structure and function of Gbetagamma as well as discuss emerging mechanisms for regulation of Gbetagamma signaling. Recent data suggest that Gbetagamma is a potential therapeutic drug target. Thus, a thorough understanding of the molecular and physiological functions of Gbetagamma has significant implications.     

Structure and assembly of the 20S proteasome

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain α-type subunits, the inner ones comprise β-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the α-type and β-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the β-type subunits are processed. The prosequence of the β-type subunits is essential for the assembly process and prevents protease activity of immature proteasomes.     

A further study of LDH isozymes in theRana esculenta complex

Summary Electrophoretic analyses of LDH isozymes in the 3 types of European green frogs by using the buffer system of Williams and Reisfeld demonstrated the occurrence of 4 allelic genes coding for the B subunits. Based on the distribution of these alleles and results from dissociation and recombination of the subunits, the hybrid nature of Rana esculenta is confirmed.This study was supported in part by the Swiss National Science Foundation and the Georges-und-Antoine-Claraz-Schenkung.     

Assessing heterogeneity in oligomeric AAA+ machines

ATPases Associated with various cellular Activities (AAA+ ATPases) are molecular motors that use the energy of ATP binding and hydrolysis to remodel their target macromolecules. The majority of these ATPases form ring-shaped hexamers in which the active sites are located at the interfaces between neighboring subunits. Structural changes initiate in an active site and propagate to distant motor parts that interface and reshape the target macromolecules, thereby performing mechanical work. During the functioning cycle, the AAA+ motor transits through multiple distinct states. Ring architecture and placement of the catalytic sites at the intersubunit interfaces allow for a unique level of coordination among subunits of the motor. This in turn results in conformational differences among subunits and overall asymmetry of the motor ring as it functions. To date, a large amount of structural information has been gathered for different AAA+ motors, but even for the most characterized of them only a few structural states are known and the full mechanistic cycle cannot be yet reconstructed. Therefore, the first part of this work will provide a broad overview of what arrangements of AAA+ subunits have been structurally observed focusing on diversity of ATPase oligomeric ensembles and heterogeneity within the ensembles. The second part of this review will concentrate on methods that assess structural and functional heterogeneity among subunits of AAA+ motors, thus bringing us closer to understanding the mechanism of these fascinating molecular motors.     

Isolation and characterization of hemagglutinins from the sponge Dysidea herbacea

The sponge Dysidea herbacea (Keller) was found to possess hemagglutinins. The major component, DHA-I, is a protein with a mol. wt of 26,000, which dissociates into subunits of equal size (14,000). It contains large amounts of glutamic acid and aspartic acid residues, but no half-cystine, methionine or histidine residues. DHA-I reacted with rabbit and human AB0 erythrocytes. D-galactose and lactose were effective inhibitors of DHA-I. The sponge also contained a minor component(s) which reacted preferentially with rabbit erythrocytes but not with human AB0 erythrocytes.