The principal islet of the coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.     

The role of Arg-96 in Danio rerio creatine kinase in substrate recognition and active center configuration

In creatine kinases (CKs), the amino acid residue-96 is a strictly conserved arginine. This residue is not directly associated with substrate binding, but it is located close to the binding site of the substrate creatine. On the other hand, the residue-96 is known to be involved in expression in the substrate specificity of various other phosphagen (guanidino) kinases, since each enzyme has a specific residue at this position: arginine kinase (Tyr), glycocyamine kinase (Ile), taurocyamine kinase (His) and lombricine kinase (Lys). To gain a greater understanding of the role of residue-96 in CKs, we replaced this residue in zebra fish Danio rerio cytoplasmic CK with other 19 amino acids, and expressed these constructs in Escherichia coli. All the twenty recombinant enzymes, including the wild-type, were obtained as soluble form, and their activities were determined in the forward direction. Compared with the activity of wild-type, the R96K mutant showed significant activity (8.3% to the wild-type), but 10 mutants (R96Y, A, S, E, H, T, F, C, V and N) showed a weak activity (0.056–1.0%). In the remaining mutants (R96Q, G, M, P, L, W, D and I), the activity was less than 0.05%. Our mutagenesis studies indicated that Arg-96 in Danio CK can be substituted for partially by Lys, but other replacements caused remarkable loss of activity. From careful inspection of the crystal structures (transition state analog complex (TSAC) and open state) of Torpedo cytoplasmic CK, we found that the side chain of R96 forms hydrogen bonds with A339 and D340 only in the TSAC structure. Based on the assumption that CKs consist of four dynamic domains (domains 1–3, and fixed domain), the above hydrogen bonds act to link putative domains 1 and 3 in TSAC structure. We suggest that residue-96 in CK and equivalent residues in other phosphagen kinases, which are structurally similar, have dual roles: (1) one involves in distinguishing guanidino substrates, and (2) the other plays a key role in organizing the hydrogen-bond network around residue-96 which offers an appropriate active center for the high catalytic turnover. The mode of development of the network appears to be unique each phosphagen kinase, reflecting evolution of each enzyme.     

The temperature dependence of creatine kinase fluxes in the rat heart

A ³¹P nuclear magnetic resonance saturation transfer method was used to measure the temperature dependence of creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts. A decrease in temperature from 37 to 4°C lowered the observed steady-state fluxes by about 80%. These data were used in conjunction with calculated changes in substrate concentrations with temperature to estimate the activation energy for creatine kinase in situ. The apparent activation energy of 42 kJ/mol agrees reasonably well with the range of literature values for the enzyme in vitro. This demonstrates that the reaction is not diffusion-limited in situ and that extraction and dilution of the enzyme for study in vitro does not alter fundamental kinetic properties of the enzyme exhibited in the intact tissue.     

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 μmol/min/mg for cytosolic CK and 240 μmol/min/mg for MtCK. Native M_rs of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.     

Quantitative determination of creatine kinase release from herring (Clupea harengus) spermatozoa induced by tributyltin

Creatine kinase (CK, ATP creatine phosphotransferase, EC 2.7.3.2) is an enzyme participating in ATP regeneration, which is the primary source of energy in living organisms. We demonstrated that CK from herring spermatozoa has high activity ( approximately 452 micromol/min/g of fresh semen) and has a different electrophoretic mobility from isoenzymes present in skeletal muscle. In our study, we investigated toxic effect of tributyltin (TBT) on herring spermatozoa using a specific sperm viability kit to observe live and dead sperm cells with a confocal microscope. Treatment of herring spermatozoa with TBT caused a time-dependent decrease of viability: 35% nonviable cells with 5 microM TBT and more than 90% nonviable cells with 10 microM TBT after 6 h exposure. We also monitored CK release from damaged spermatozoa into surrounding medium containing different concentrations of TBT. The higher concentration of TBT was used the more CK release from spermatozoa was observed. We suggest that CK could be a good biomarker of sperm cell membranes degradation in the case when lactate dehydrogenase release from permeabilized cells is not possible for rapid determination of the effect of TBT.     

Estimation of protein digestibility—IV. Digestive proteinases from the pyloric caeca of coho salmon (Oncorhynchus kisutch) fed diets containing soybean meal

The goal of this study was to better understand why dietary soybean products are poorly utilized by salmonids. The influence of dietary intake on coho salmon fingerling weight gain and specific properties of pyloric caeca enzymes was investigated. Fingerlings were fed diets containing heated or unheated soybean meal (SBM) or Promoveal™, as 15–25% herring meal replacer, for 8–12 weeks. Fish fed to apparent satiation with diets containing heated SBM replacer gained more weight than those fed unheated SBM at the same level. Fish increased in body weight at the same rate when fed restricted rations containing either 15% SBM replacer that was variously heated up to 20 min, 15% Promoveal™ replacer or the herring meal basal diet. After the experimental diets were fed, digestive proteinases were isolated from the pyloric caeca. Yield of pyloric caeca enzymes (PCE), recovery of trypsin in PCE, soybean trypsin inhibitor (SBTI) sensitivity of PCE trypsin, specific activity of PCE trypsin and in vitro casein digestibility by PCE were determined for each dietary group. Weight gain vs in vitro casein digestibility by PCE was linear for animals fed unheated SBM to apparent satiation (r² = 0.71, P < 0.1) but not for animals fed either heated SBM to apparent satiation or variously heated SBM as 15% replacer at restricted levels. Trypsin from fish fed diets with heated or unheated SBM, but not Promoveal™ replacer, was less sensitive to SBTI than fish fed no SBM. For fish fed diets with variously heated SBM as 15% replacer, the SBTI activity of the SBM and SBTI inhibition of PCE trypsin were inversely related (r² = 0.88, P < 0.05). The yield of PCE was higher for fish fed 25% of heated SBM replacer than it was for diet groups fed less SBM. The yield of PCE trypsin was higher from animals fed 25% heated SBM replacer than those fed diets with a lower percentage of heated SBM replacer. Feeding coho fingerlings rations with SBM replacer appears to promote physiological compensation of PCE. Heat stable and/or heat-activated factor(s) and SBTI appear to cause the compensation of salmon digestive proteinases from coho salmon fed diets with SBM.     

The endocrine pancreas in the adult gray short-tailed opossum, Monodelphis domestica (Marsupialia)

Monodelphis domestica, a South American marsupial proposed as a laboratory animal, presents some characteristics that are favourable for research on the development of pancreatic tissue. Therefore, it is important to know if Monodelphis domestica is similar to other mammals concerning the cellular composition of the pancreas and if the different regions of the organ share similar morphologic features. In the present study the pancreas of ten adult male or female Monodelphis domestica were examined: serial paraffin sections were stained immunohistochemically using polyclonal antibodies against insulin or glucagon. The size of the immunoreactive area was measured with a computer-based image-analysing system differentiating lobus dexter, corpus pancreatis, and lobus sinister.

The islets' profiles varied in size and shape. The insulin-positive cells were mainly located in the centre of the islets, while the glucagon-positive cells were arranged at the islets' periphery. However, some glucagon-positive cells also occurred in the centre of most islets. On average, 0.66% of the entire pancreatic section area was insulin-positive, while 0.23% was glucagon-positive. Thus, the ratio between glucagon-positive and insulin-positive area was 1:2.9. Differences between the three pancreatic parts, lobus dexter, corpus pancreatis, and lobus sinister, were revealed. In general, the findings compared well with those of species that have a history as laboratory animals, i.e. mice, rats, cats, and dogs.     

Genetic heterogeneity within populations of Lepeophtheirus europaensis (Copepoda: Caligidae) parasitic on two host species

, and 1992. Genetic heterogeneity within populations of Lepeophtheirus europaensis (Copepoda: Caligidae) parasitic on two host species. International Journal for Parasitology 22: 1179–1181. The ectoparasitic copepod Lepeophtheirus europaensis exploits two different species of Mediterranean flat fishes: the brill (a marine fish) and the flounder (found in lagoons). Analysis of the genetic structure of these parasite populations revealed that migration is reduced between the two hosts. Moreover, an adaptive polymorphism was found when the survival of the eggs and free larvae of these copepods was studied at different salinities. The results are discussed and interpreted as signs of a probable speciation process currently occurring within this parasite taxon.     

银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。     

The pancreatic islet system of the mouse (Mus musculus)

Summary The pancreatic islet in the mouse has a highly complex and heterogeneous structure. It contains Aa, Ab, Ac, B, C, D, E, and F cells. The classification of cell types is primarily based on the shape, size and electron opacity of secretory granules and on the spatial relationship of the granules to their unit membranes. Morphological evidence is supported by a statistical analysis of the size distribution of granules and of their membranes. Experimental immunization of mice with insulin, provides additional data to support the existence of eight different cell types in the islet of the normal animal and reveales marked immunological stimulation of B cells, secondary stimulation of Aa, D and F cells, atrophy of Ac cells and hyperplasia of C cells. It is proposed that corresponding cell types exist in other mammals and man. The experimental insulin immunization process appears to perform an immunofunctional analysis of the islet, and suggests that in mice the Aa, D and F cells might be involved in cell energy supply. Lipocaic and some pancreatic factors with insulin-like activity (NSILA) will likely find their morphological equivalents. It is proposed that chemical solubility techniques represent the most promising avenues of approach to the isolation of secretory products from the endocrine pancreas, and that the assay of these extracts should primarily be conducted at the cell level.Dedicated to Prof. Dr. med. W. Masshoff on his 60th birthday.The author is indebted to Dr. med. H. J. Stolpmann for guidance in applying the techniques of electron microscopy and wishes to express his special appreciation to Mrs. Marjanne Hinz for her valuable assistance in completing different aspects of this work and for her competent technical aid.     

大叶落地生根类糖原合成激酶KdSK基因的克隆与表达分析

类糖原合成酶激酶(SKs)属于丝氨酸/苏氨酸类蛋白激酶,在植物器官发育、激素信号传导过程中十分重要,并参与生物胁迫、非生物胁迫的应答过程。大叶落地生根中的胎生苗发育过程,同时具备胚胎发生和器官发生的特征,是研究无性生殖的理想模型。为了更好地理解大叶落地生根中胎生苗发育的分子机制,该研究利用RACE-PCR技术,从大叶落地生根中克隆了1个新的基因KdSK。该基因具有423个氨基酸残基,分子量为47.79 kD,等电点为8.37,其开放阅读框长为1 272 bp。其蛋白与黄瓜的同源性最高,属于植物类GSK3/shaggy蛋白激酶家族的第Ⅳ类,与苜蓿(MSK4)蛋白在进化关系上最近,且与拟南芥(AtSK4-1、AtKSK4-2)聚为一枝。保守域结构分析表明,KdSK蛋白具有明显的蛋白激酶的结构域,包括蛋白激酶的ATP结构域和丝氨酸/苏氨酸蛋白激酶活化结构域。实时荧光定量PCR分析表明,该蛋白基因在大叶落地生根的根中表达量最高,且受渗透胁迫(甘露醇)的诱导上调表达。该研究首次从大叶落地生根中克隆出KdSK基因,该研究结果为进一步研究该基因的功能打下了基础。     

Activation of protein kinase isoenzymes under near physiological conditions. Evidence that both types (A and B) of cAMP binding sites are involved in the activation of protein kinase by cAMP and 8-N3-cAMP

cAMP-dependent protein kinase I and II (cAKI and cAKII) were incubated under near physiological conditions in the presence of various concentrations of 8-N3-c[3H]AMP or c[3H]AMP. Both types (A and B) of cyclic nucleotide binding sites of cAKI or cAKII were occupied to a similar extent and the degree of their occupation correlated with the degree of kinase activation. cAKI and cAKII bound cAMP in an apparent positively cooperative manner in the presence of Mg2+, ATP. 8-N3-c[3H]AMP dissociated several orders of magnitude faster from site A than site B of the regulatory moiety of cAKII, and was photo-incorporated only when bound to site B.     

A 31P-NMR saturation transfer study of the regulation of creatine kinase in the rat heart

(1) ³¹P nuclear magnetic resonance was used to measure the creatine kinase-catalysed fluxes in Langendorff-perfused rat hearts consuming oxygen at different rates and using either of two exogenous substrates (11 mM glucose or 5 mM acetate). (2) Fluxes in the direction of ATP synthesis were between 3.5–12-times the steady-state rates of ATP utilization (estimated from rates of O₂-consumption), demonstrating that the reaction is sufficiently rapid to maintain the cytosolic reactants near their equilibrium concentrations. (3) Under all conditions studied, the cytosolic free [ADP] was primarily responsible for regulating the creatine kinase fluxes. The enzyme displayed a K_m for cytosolic ADP of 35 μM and an apparent V_max of 5.5 mM/s in the intact tissue. (4) Although the reaction is maintained in an overall steady-state, the measured ratio of the forward flux (ATP synthesis) to the reverse flux (phosphocreatine synthesis) was significantly greater than unity under some conditions. It is proposed that this discrepancy may be a consequence of participation of ATP in reactions other than the PCr /ag ATP or ATP /ag ADP + P_i interconversions specifically considered in the analysis. (5) The results support the view that creatine kinase functions primarily to maintain low cytosolic concentrations of ADP during transient periods in which energy utilization exceeds production.     

Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps

Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of lysine residues that are highly conserved in M-CK but are not present in B-CK. The role of these lysines in mediating M-line interaction was tested with a set of M-CK and B-CK point mutants and chimeras. We found that all four lysine residues are involved in the isoenzyme-specific M-line interaction, acting pair-wise as strong (K104/K115) and weak interaction sites (K8/K24). An exchange of these lysines in MM-CK led to a loss of M-line binding, whereas the introduction of the very same lysines into BB-CK led to a gain of function by transforming BB-CK into a fully competent M-line-binding protein. The role of the four lysines in MM-CK is discussed within the context of the recently solved x-ray structures of MM-CK and BB-CK.     

The influence of temperature on catalytic efficiency of pyruvate kinase of crickets (Orthoptera: gryllidae)

Investigations have been conducted to determine the chemical nature of immediate temperature-regulatory mechanisms for enzyme activity, such as positive or negative temperature modulation and an adaptation-temperature dependence of the free energy of activation ΔG^≠. Three species of crickets have been selected for experiments in consideration of their different natural temperature demands: Gryllus campestris, Gryllus bimaculatus, and Acheta domesticus. Discontinuous Arrhenius plots (Fig. 1) show that all pyruvate kinases can exist in at least two temperature-dependent conformational states. Sizes of ΔH^≠-and ΔG^≠-values are correlated with the species' adaptation temperature (Table 1). Decreased barriers of ΔG^≠ after cold adaptation in G. campestris and A. domesticus are not sufficient for complete temperature compensation of the catalytic efficiency. Maximum enzyme-substrate affinity closely corresponds to the acclimation temperature of the crickets (Fig.2); K_m-values for PEP, however, are hardly influenced by experimental temperatures within the normal temperature range of the species. Data on enzyme function appear to corroborate the idea that optimal catalytic properties will be set according to the highest temperatures experienced respectively.     

Isoforms of Glutamine Synthetase in the Marine Coccolithophorid Emiliania huxleyi (Prymnesiophyceae)

Two isoenzymes of glutamine synthetase (EC 6.3.1.2), GS₁ and GS₂, have been purified from cells of Emiliania huxleyi using Cibacron blue dye ligand chromatography and gel filtration, separated by ion-exchange chromatography on Mono-Q and partly characterized. Each enzyme is a homohexamer with a molecular mass of 402 kDa for GS₁ and 501 kDa for GS₂. The molecular mass of the subunits of GS₁ and GS₂ was estimated to be 61 and 78 kDa, respectively. As in higher plants, GS₁ is slightly more thermostable than GS₂ and much less stimulated by thiols than GS₂. For these reasons, GS₁ was designated as the cytosolic enzyme and GS₂ as the chloroplastic one. Although the K_ms for NH₂OH are about the same, GS₂ possesses a much higher affinity for glutamine than GS₁. As in bacteria, ATP appears to play an important role in the allosteric regulation of GS₂. l-Ala and CTP are potent inhibitors of GS₁ activity. CTP, carbamoyl-phosphate and l-Ala exert a cumulative inhibitory effect on GS₁ activity. GS₂ is also inhibited to some extent by l-Ala and l-His. NH₂-terminal sequence analysis of GS₂ did not show any homology with bacteria, cyanobacteria or higher plants.     

Relationship between the aerobic and anaerobic metabolic capacities and the vertical distribution of three intertidal sessile invertebrates: Jehlius cirratus(Darwin) (Cirripedia), Perumytilus purpuratus (Lamarck) (Bivalvia) and Mytilus chilensis (Hupé) (Bivalvia)

In protected areas of the southern Chilean coastline, the barnacle Jehlius cirratus dominates the upper intertidal zone, the bivalve Perumytilus purpuratus is the dominating species in the intermediate zone and Mytilus chilensis in the middle-lower zone. Varying degrees of aerial respiration were observed in the laboratory for these three sessile invertebrates: 80–100% of the immersed oxygen uptake for Jehlius cirratus, 30–50% for Perumytilus purpuratus and 5–15% for Mytilus chilensis. The pyruvate kinase/phosphoenolpyruvate carboxykinase (PK/PEPCK) ratio (adductor muscle) was higher for P. purpuratus than for M. chilensis. J. cirratus had no PEPCK activity in total soft tissues, under the assay conditions used in this study, and PK activity was higher for the cirriped than that of both bivalves. Succinate was produced in both mussels when exposed to hypoxic conditions, but it was not detected in J. cirratus. The kinetic parameters of the PK revealed that M. chilensis presented the highest _0.5 value for PEP and J. cirratus the lowest. Alanine inhibited the PK from M. chilensis to a greater extent (higher n_H value) and did not affect the PK of the cirriped. This latter enzyme also presented a marked substrate inhibition, above PEP concentrations of 0.5 mM. The evidence suggests that organisms inhabiting the upper intertidal zone could utilize an aerobic metabolism, given their high aerial respiration capacity, and that the succinate anaerobic pathway could be a more important metabolic strategy in species of the middle-lower intertidal zone. The physiological and biochemical capacities of these three species could thus be correlated with their vertical distribution in the intertidal rocky shore.