Production of thermostable amylolytic enzymes by Thermococcus hydrothermalis

A cell extract of Thermococcus hydrothermalis, grown for 6 h, gave -glucosidase activity at 14.9 U/l, degrading oligosaccharides and maltose. -Amylase, -glucosidase and pullulanase activities were detected at 289 U/l, 13.5 U/l and 30 U/l respectively in the culture medium after 24 h growth of the archaeum. All of three enzymes, characterised by a half-life time of 1 to 5 h at 95°C, degraded both the (14) and (16) linkages of polysaccharides and the (14) linkages of oligosaccharides. © Rapid Science Ltd. 1998     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

The influence of nearest neighbors on the rate and pattern of spontaneous point mutations

Summary The numbers and local sequence environments of the two types of substitution mutation plus additions and deletions have been obtained directly in this study from differences between a large number of extant primate gene and pseudogene sequences. A total of 3786 mutations were scored in regions where similarities between pseudogene and corresponding gene sequences is 85%, comprising 30% of the pseudogene database of 80,584 bp. The pattern of mutations obtained in this fashion is almost identical to that obtained by Li et al. (1984) using a slightly different, more direct approach and with a smaller database. When mutations were scored, the neighbor pairs on the 5 and 3 sides were also noted, leading to a large 16 × 12 matrix of transitions and transversions. Biases of varying magnitude are found in the rates of substitution of the same base pair in different local sequence environments. The overall order for the effect of the 5 neighbor on the rates of substitution mutation of a pyrimidine is A > C T > G, and G > A > T > C for the 3 neighbor; where these results represent the average of substitution rates for the complement purine with complement neighbors of bases ordered above. The order for the 3 neighbor is essentially the same for the two transitions and most of the four transversions as well; however, the order for the 5 neighbor is more variable. The overall rate for the C · G T · A transition is not unusual, however the presence of a 3 neighboring G · C pair boosts the rate substantially, presumably due to specific cytosine methylation of the CG doublet in primate DNAs. The rate of the T · A C · G transition is also well above average when the 3 neighbor is an A · T, and to a lesser extent a G · C, pair. The latter bias is typical in that it reflects the association of alternating pyrimidine-purine sequences with increasing mutation rates. The substitution of the pyrimidine in a 5 purine-pyrimi-dine-purine3 sequence generally occurs much faster than in a pyrimidine tract and points to the local conformation as a major determining factor of the substitution rate. An apparent inverse relationship is found between starting and product doublet frequencies of base pairs undergoing mutations with specific 3 neighbors, indicating that differences in intrinsic substitution rates of base pairs with specific neighbors are a key factor in producing the familiar biases of nearest-neighbor frequencies.Offprint requests to: R. D. Blake     

Novel Type of Signaling Molecules: Protein Kinases Covalently Linked with Ion Channels

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyosteliummyosin heavy chain kinases. Here we cloned, sequenced, and analyzed the tissue distribution of five new putative mammalian -kinases: melanoma -kinase, kidney -kinase, heart -kinase, skeletal muscle -kinase, and lymphocyte -kinase. All five are large proteins of more than 1000 amino acids with an -kinase catalytic domain located in the carboxyterminal part. We expressed the catalytic domain of melanoma -kinase in Escherichia coli, and found that it autophosphorylates at threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that long aminoterminal portions of melanoma and kidney -kinases represent new members of the TRP ion channel family, which are thought to mediate the capacitative Ca²⁺entry in nonexcitable mammalian cells. This suggests that melanoma and kidney -kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca²⁺influx in mammalian cells.     

An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins

Summary A 3D NMR technique is described which correlates the amide proton and nitrogen resonances of an amino acid residue with the C chemical shift of its preceding residue. The technique uses a relay mechanism, transferring magnetization from¹⁵N to¹³C via the intervening carbonyl nucleus. This method for obtaining sequential connectivity is less sensitive to large line widths than the alternative HNCA experiment. The technique is demonstrated for the protein calmodulin, complexed with a 26 amino acid fragment of skeletal muscle myosin light chain kinase.Abbreviations CaM Calmodulin - HCACO -proton to -carbon to carbonyl correlation - H(CA)NHN -proton (via -carbon) to nitrogen to amide proton correlation - HMQC heteronuclear multiple quantum correlation - HNCA amide proton to nitrogen to C -carbon correlation - M13 a 26-residue fragment of the CaM-binding domain of skeletal muscle myosin light chain kinase comprising residues 577–602.     

Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Double-immunofluorescent staining of isolated smooth muscle cells

Summary Contractile proteins have been co-localized by double-immunofluorescent staining in several types of cultured cells. Since freshly isolated smooth muscle cells are more representative of the organization within smooth muscle cells in the intact tissue than cultured cells, the present study was undertaken to determine the feasibility of using double-staining techniques in freshly isolated cells. A new method of purifying -actinin from chicken gizzards was used to provide antigen for raising anti--actinin. Fluorescein isothiocyanate-labelled anti--actinin (FAA) was used in conjunction with tetramethyl rhodamine isothiocyanate-labelled anti-myosin (TRAM) Ouchterlony gels against myosin, tropomyosin, actin, and -actinin showed that antimyosin reacted only with myosin, anti--actinin only with -actinin. Anti--actinin stained only the Z-line of isolated chicken skeletal muscle myofibrils. FAA stained bright, discrete patches or strips on the plasma membrane, while TRAM was excluded from these areas. FAA stained myofibrils faintly in a striated pattern, while TRAM stained myofibrils heavily with less evident striations. Evidence for extramyofibrillar localization of -actinin within the cytoplasm was inconclusive. Although antibodies were quite specific in their labelling, resolution with double-staining was subject to the same limitations described for single labelling of whole cells (Bagby and Pepe 1978). Double-staining of whole cells is just as feasible as single-staining. Indeed, having a definite marker for myofibrils (TRAM) makes the localization of -actinin much easier to interpret.     

Phylogenetic relationship of zeins and coixins as determined by immunological cross-reactivity and Southern blot analysis

Zeins from Zea mays L cv. Maya and coixins from Coix lacryma-jobi L. cv. Adlay were fractionated to obtain -, -, and -zein and -, -, and -coixin. The -coixins were composed of 4 polypeptide classes of 27 kDa (C1), 25 kDa (C2), 17 kDa (C4) and 15 kDa (C5) with solubility properties very similar to those of the 22 kDa and 19 kDa -zeins. Like the -zeins, the C1 and C2 -coixins corresponded to 80% of total Coix prolamins. The fraction corresponding to -coixin contained only one protein band of 22 kDa (C3). This coixin fraction has solubility properties similar to those of -zein and represents 15% of the total coixin. The -zein fraction was composed of a major 17 kDa protein band, while the -coixin fraction consisted of a mixture of - and -coixins.Polyclonal antibodies raised against C1 recognized C1 and C2 and cross-reacted strongly with the 22 kDa -zein, as did C4 and C5 antisera. The antiserum against -coixin showed strong cross-reaction with -zein. The homology between coixins and zeins was further investigated by using Southern hybridization analyses. The genomic DNA of maize and Coix were digested with several restriction enzymes and probed with cDNA clones representing 19 and 22 kDa -zeins as well as the 28 and 16 kDa -zeins. The Coix genome showed complex cross-hybridization sequences with the 22 kDa -zein cDNA, while no cross-hybridization was observed with the 19 kDa cDNA clone. The cDNA clone representing the 28 kDa -zein cross-hybridized with only one band of Coix genomic DNA, in contrast to the three bands observed in maize. This same Coix sequence also cross-hybridized with the cDNA clone representing the 16 kDa -zein. The relevance of these findings are discussed in the context of the origin of zein and coixin genes.     

Demonstration of ‘cardiac-specific’ myosin heavy chain in masticatory muscles of human and rabbit

Summary Human and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to cardiac , slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle cardiac myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express cardiac myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of cardiac myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The cardiac myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.     

The Expression of Transforming Growth Factor Alpha Receptor Protein and its Activation in Chicken Ovarian Granulosa Cells of Maturing Follicles

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-. In a previous study, we localized EGF/TGF- receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF- binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF- receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF- followed by crosslinking with bis(sulphosuccinimidyl)suberate showed that TGF- binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF- to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF--stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF=TGF- receptor protein, which dimerizes on binding to TGF-, suggesting that the receptor protein may be involved in the signal transduction of TGF- actions in the chicken granulosa cells.     

Acclimation of barley to changes in light intensity: photosynthetic electron transport activity and components

Barley seedlings (Hordeum vulgare L. Boone) were grown at 20°C with 16 h/8 h light/dark cycle of either high (H) intensity (500 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) and low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod. Thylakoid membranes were isolated from 3 cm apical segments and assayed for photosynthetic electron transport, Photosystem II (PS II) atrazine-binding sites (Q_B), cytochrome f(Cytf) and the P-700 reaction center of Photosystem I (PS I). Whole chain, PS I and PS II electron transport activities were higher in H than in L controls. Q_B and Cytf were elevated in H plants compared with L plants. The acclimation of H L plants to low light occurred slowly over a period of 7 days and resulted in decreased whole chain and PS II electron transport with variable effects on PS I activity. The decrease in electron transport of H L plants was associated with a decrease in both Q_B and Cytf. In L H plants, acclimation to high light occurred slowly over a period of 7 days with increased whole chain, PS I and PS II activities. The increase in L H electron transport was associated with increased levels of Q_B and Cytf. In contrast to the light intensity effects on Q_B levels, the P-700 content was similar in both control and transferred plants. Therefore, PS II/PS I ratios were dependent on light environment.Abbreviations Asc ascorbate - BQ 2,5-dimethyl-p-benzoquinone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L low control plants grown under low light intensity - L H plants transferred from low to high light intensity - MV methyl viologen - P-700 photoreaction center of Photosystem I - Q_B atrazine binding site - TMPD N,N,N,N-tetramethyl-p-phenylenediamine Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11990 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature

Summary The histochemical activities of succinic dehydrogenase (SDH) and Ca⁺⁺-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. Red and white muscle fibres, as distinguished by SDH, showed high and low Ca⁺⁺-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K₂-EDTA solution. The ultrastructural investigation of CA⁺⁺-ATPase reaction at pH 7.4 by the Ca⁺⁺-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and Z bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb⁺⁺ precipitation technique demonstrated Mg⁺⁺-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail red muscle fibres are possible slow, and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, red muscle fibres of the anuran tail musculature are not equivalent to Type I fibres of higher chordates.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Partial purification of myosin from lily pollen tubes by monitoring with in vitro motility assay

Summary Myosin in pollen tubes ofLilium longiflorum was partially purified, using an in vitro motility assay as a monitor. The main components in the partially purified preparation had molecular masses of 110, 120, and 140 kDa in SDS-PAGE. They became bound to actin filaments in an ATP-dependent manner. Among the components, only that of 120 kDa became bound to ATP and was concluded to be the heavy chain of pollen tube myosin.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EB extraction buffer - EGTA ethyleneglycol-bis-(-aminoethylether) N, N, N, N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - TBS Tris buffered saline - TEB Tris-EGTA buffer     

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum by PKCα, PKCβ, PKA and the Ca2+/calmodulin-dependent protein kinase

Phosphorylation of cardiac junctional and free sarcoplasmic reticulum (SR) by protein kinase C (PKC) isoforms and was investigated. Both SR and PKC were isolated from canine heart. Junctional and free SR vesicles were prepared by calcium-phosphate-loading. The substrate specificities of PKC and PKC were found to be similar in both SR fractions. A high molecular weight junctionally-associated protein was phosphorylated by PKA, PKC and an endogenous Ca²⁺/calmodulin-dependent protein kinase activity: the highest levels of phosphate incorporation being catalysed by the latter kinase. In addition to this high molecular weight junctionally-associated protein, PKC induced phosphorylation of 45, 96 kDa and several proteins of greater than 200 kDa in junctional SR. A protein of 96 kDa was phosphorylated by both isoforms in junctional and free SR. The major substrate for PKA, PKC, PKC and the Ca²⁺/calmodulin-dependent protein kinase, in both junctional and free SR, was phospholamban. Although the phosphorylation of phospholamban by PKC was activated by Ca²⁺, a component of this activity appeared to be independent of Ca²⁺. PKC-mediated phosphorylation of phospholamban was fully activated by 1 M Ca²⁺ whereas the Ca²⁺/calmodulin dependent kinase required concentrations in excess of 5 M Ca²⁺. In the in vitro system employed in these studies, the concentrations of either PKC or the catalytic subunit of PKA required to phosphorylate phospholamban were found to be similar. In addition, in the presence of a 15 kDa sarcolemmal-associated protein, which becomes phosphorylated upon activation of PKC in vivo, phosphorylation of phospholamban by PKC was unaffected. These results demonstrate that, although substrates for both subtypes are found in both junctional and free SR, PKC and PKC do not show differences in selectivity towards these substrates.Abbreviations Ca²⁺ free calcium - CaM kinase Ca²⁺/calmodulin-dependent protein kinase - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis(b-aminoethylether)-N,N,N,N-tetraacetic acid - FSR free sarcoplasmic reticulum - JSR junctional sarcoplasmic reticulum - PKC protein kinase C - PS phosphatidylserine - SDS sodium dodecyl sulfate - SAG 1-stearoyl-2-arachidonylglycerol - TPCK L-1-tosylamido-2-phenylethyl chloromethyl ketone - Tris/HCI tris(hydroxymethyl)aminomethane hydrochloride This work was supported by a grant (to S.K.) from the Heart and Stroke Foundation of B.C. and Yukon. The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Recipient of a Studentship form the Heart and Stroke Foundation of Canada.     

Isolation and characterization of plant myosin from pollen tubes of lily

Summary A plant myosin was isolated from pollen tubes of lily,Lilium longiflorum. Pollen tubes were homogenized in low ionic strength solution containing casein, and myosin from this crude extract was purified by co-precipitation with F-actin prepared from chicken breast muscle, followed by hydroxylapatite column and gel filtration column chromatography. Upon SDS-PAGE on 6% polyacrylamide gel, only 170 kDa polypeptide was detected in the purified myosin fraction. Furthermore, with immunoblotting using antiserum raised against 170 kDa polypeptide, only the 170 kDa component crossreacted in the crude sample of pollen tube proteins. This antiserum did not crossreact with the heavy chain of skeletal muscle myosin. The ATPase activity of pollen tube myosin was stimulated up to 60-fold by F-actin prepared from chicken breast muscle. The translocation velocity of rhodamine-phalloidin-labeled F-actin on a glass surface covered with pollen tube myosin ranged from 6.0 to 9.8 m/s with an average of 7.7 m/s. This velocity was similar to or a little faster than that of the cytoplasmic streaming that occurred in pollen tubes. These results suggested that myosin composed of a 170 kDa heavy chain produces the motive force for cytoplasmic streaming in pollen tube of lily.Abbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazin-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate     

Zur Histochemie von Hydrolasen im Hoden des Hundes und der Katze

Zusammenfassung Wir untersuchten das Verteilungsmuster von unspezifischer Esterase, alkalischer Phosphatase, Adenosintriphosphatase, 5-Nucleotidase und -D-Glucuronidase im Hoden von Hund und Katze. Besonders hervorzuheben sind eine starke Aktivität der unspezifischen Esterase in den Sertolizellen der Katze, der Reichtum der Membrana propria aller Hodentubuli an alkalischer Phosphatase und Adenosintriphosphatase sowie die kräftige Reaktion auf -D-Glucuronidase in den Leydigzellen beider Tierarten.Die Befunde werden diskutiert.

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Summary The localization of unspecific esterase, alkaline phosphatase, adenosine triphosphatase, 5-nucleotidase, and -D-glucuronidase in the testes of cat and dog was demonstrated by histochemical means. We observed a strong esterase activity in the Sertoli cells of the cat and high amounts of alkaline phosphatase and adenosine triphosphatase in the membrana propria of all seminiferous tubules. In both species the principal site of -D-glucuronidase was in the Leydig cells. Our findings obtained being discussed.

     

Subunit composition of the zinc proteins alpha- and beta-lipovitellin from chicken

Chicken - and -lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 ± 0.2 mol of zinc/275 kDa per -lipovitellin and 1.4 ± 0.2 mol of zinc/155 kDa per -lipovitellin, respectively. The subunits of -lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3–16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of - and -lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from - and -lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of - and -Lv, respectively, and two peptides of the 30-kDa subunit of - and -lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of - and -lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of -lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor—product relationship of Vtg I.     

Wheat chromosome 2D carries genes controlling the activity of two DNA-degrading enzymes

DNA-degrading enzymes of 24.0 kDa and 27.0 kDa were observed to have different activities in two common wheat (Triticum aestivum L.) cultivars, Wichita and Cheyenne. A substrate-based SDS-PAGE assay revealed that these two enzymes were much more active in Wichita than in Cheyenne. Genes controlling the activities of these two enzymes were localized on chromosome 2D by testing DNA-degrading activities in reciprocal chromosome substitution lines between Wichita and Cheyenne. While the allele on Wichita chromosome 2D stimulated the activities of the 24.0- and 27.0-kDa enzymes in Cheyenne, the allele on Cheyenne chromosome 2D did not reduce the activities of the 24-kDa and 27-kDa enzymes in Wichita. Whether these genes code for the DNA-degrading enzymes themselves or for factors that regulate the enzyme activities remains unknown.This work was supported in part by USDA-Competitive Research Grants Office grant No. 90-37140-5426 to P.S.B. Contribution from Agricultural Research Division, University of Nebraska. Journal Series Number 10304