Activation of glycogen synthetase and inactivation of phosphorylase kinase by the same phosphoprotein phosphatase

Purified rabbit muscle glycogen synthetase D phosphatase inactivates phosphorylase kinase. The inactivation is reversed by cyclic AMP-dependent protein kinase. It is postulated that the synthetase D phosphatase is a general phosphoprotein phosphatase which dephosphorylates proteins that are phosphorylated $\text{in}$$\text{vivo}$ by the cyclic AMP-dependent kinase.     

Protein phosphorylation in rod outer segments from bovine retina: cyclic nucleotide-activated protein kinase and its endogenous substrate.

Protein kinase activity of isolated rod outer segments from bovine retinas is activated by cGMP when in a soluble form, and it is cyclic nucleotide independent when associated with the rod outer segment membranes. The soluble protein kinase phosphorylates in a cyclic nucleotide-dependent manner only a single endogenous protein with an apparent molecular weight of 30,000 daltons. The 30,000-dalton phosphoprotein is localized specifically in the visual cells of the retina. It is proposed that the light-induced changes in cGMP levels that occur in rod outer segments $\text{in vivo}$ are linked by the cyclic nucleotide-dependent protein kinase to alterations in the content of the 30,000-dalton phosphoprotein.     

Purification of a protein kinase from human Namalwa cells that phosphorylates topoisomerase I

A nuclear protein kinase which phosphorylates phosphoprotein $\text{110}\text{8.4}$, recently identified as topoisomerase I, has been purified approximately 330 fold from a 10 mM Tris extract of human Namalwa cells. The kinase wás chromatographed on DEAE-Sephacel and further purified by affinity chromatography on phosvitin-Sepharose. The protein kinase exhibited a high affinity (Km = 0.3 μM) for topoisomerase I; its affinity for phosvitin was approximately 100 fold lower (Km = 25 μM).     

Substrate specificity of glycogen synthase phosphatase from bovine heart: action on phosphorylase a and histone

Glycogen synthase phosphatase has been purified from bovine heart. This preparation catalyzes conversion of synthase D into I and phosphorylase $\text{a}$ into $\text{b}$ and is able to dephosphorylate synthase D, phosphorylase $\text{a}$, active phosphorylase kinase, and phosphorylated histone and casein. The activity of phosphatase was assayed with synthase D, phosphorylase $\text{a}$, and histone as substrates after chromatography on Sephadex G-100, after sucrose gradient centrifugation, and after isoelectric focusing in a sucrose gradient. In all cases no separation of enzyme activity was observed with the above substrates. The phosphatase activity on all substrates was lost at the same rate by heat denaturation. These results indicate that this enzyme preparation contains a single phosphoprotein phosphatase which is responsible for the activity observed on the above substrates.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Phosphoprotein-phosphatase activity associated with human placental alkaline phosphatase.

Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein phosphatase activity. This was demonstrated by the dephosphorylation of ³²P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase $\text{a}$ phosphatase activity.     

Nτ- and Nπ-histidinoalanine: Naturally occurring cross-linking amino acids in calcium-binding phosphoproteins

Two isomeric amino acid cross-links, N^τ-histidinoalanine and N^π-histidinoalanine have been isolated from calcium-binding phosphoprotein particles derived from the extrapallial fluid of the estuarine clam $\text{Rangia}\text{,}\text{cuneata}$. The cross-links were identified and compared by ¹³C and ¹H NMR spectroscopy and mass spectroscopy. In the phosphoprotein particles, 6% of the amino acid residues are involved in cross-linkages. This is the first demonstration of the occurrence of the isomer N^π-histidinoalanine.     

Enxymology of human colon tumors

The biochemical strategy of human colon adenocarcinoma was studied by elucidating the enzymic programs of pyrimidine biosynthesis and degradation, glycolysis, pentose phosphate production, and galactose metabolism in normal colon mucosa and in 9 cases of primary colon adenocarcinoma. Enzymic activities were determined in the 100,000 X $\text{g}$ supernatant fluid with spectrophotometric or isotopic assays under optimum conditions yielding linear kinetics. In the human colon tumors the activities of enzymes of the $\text{de}$$\text{novo}$ pyrimidine biosynthesis, CTP synthetase, OMP decarboxylase, and orotate phosphoribosyltransferase, were increased to 348, 183, and 201% of those of normal human colon mucosa. The activities of the salvage pathway enzymes, thymidine kinase, uracil phosphoribosyltransferase and uridine kinase, were increased to 331, 254 and 281%. By contrast, the activity of the catabolic enzyme, uridine phosphorylase, was decreased to 69%. The ratio of activities of uridine kinase/ uridine phosphorylase was elevated to 564%. The activities of the key glycolytic enzymes, hexokinase and pyruvate kinase, and those of pentose phosphate production, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transaldolase, increased to 348, 209, 262, 156, and 180% respectively. The activity of the first committed enzyme of galactose utilization, galactokinase, was increased to 315%. The enzymic program of human primary colonic adenocarcinoma was similar in most respects to that which we observed in chemically-induced, transplantable adenocarcinomas of the colon in mouse and in rat (4). The reprogramming of gene expression in human colon tumor provides an increased capacity for biosynthesis of pyrimidines and ribose 5-phosphate, and for utilization of the glycolytic pathway and of galactose. These alterations in gene expression should confer selective advantages to the human colon tumor cells. The marked elevations in the activities of the salvage enzymes, uridine-cytidine kinase and thymidine kinase, explain in part the failure to obtain good therapeutic results with inhibitors of the $\text{de}$$\text{novo}$ pathway and account, in part at least, for the clinical difficulties encountered in the treatment of colon tumors. The elevated activities of CTP synthetase, OMP decarboxylase, uridine-cytidine kinase and thymidine kinase mark out these enzymes as targets for combination chemotherapy. Through such enzyme-pattern-targeted chemotherapy the drug treatment of human colon tumors should be improved.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Separation of heme regulated protein kinase into a high and a low molecular weight species

Protein synthesis in rabbit reticulocyte lysates is regulated by heme. In heme deficiency, a heme regulated protein kinase (HRI) is activated that phosphorylates initiation factor eIF-2. Consequently, eIF-2 is inactivated. Results described in this report show that HRI exists in crude and highly purified preparations in two forms; a high molecular weight component which sediments at a sedimentation co-efficient of 14–15S and a previously described 5.8S component (Ranu, R. S. and London, I. M. (1976) $\text{Proc}\text{.}\text{Natl}\text{.}\text{Acad}\text{.}\text{Sci}\text{.}\text{USA}$ 73, 4349–4353). The 14–15S HRI selfphosphorylates poorly and undergoes dissociation into the 5.8S component via an intermediate of 8.5–9S. The 5.8S HRI, on weight basis, is about 5–10 times more active than the 14–15S HRI. In addition, a phosphoprotein phosphatase has been detected in lysates that dephosphorylates selfphosphorylated HRI. This observation suggests that phosphate on HRI turns over. These findings may be relevant ot the mechanism of activation and inactivation of HRI in the absence and presence of heme $\text{in}\text{situ}$.     

Autonomy for a specific gene product in oocytes: Experimental evidence in the polychaetous annelid,Platynereis dumerilii

Oocytes of Platynereis dumerilii in early vitellogenesis were injected into female worms with oocytes of similar diameter. The donor oocytes were labeled by the or gene controlling eye pigmentation and, after some weeks of growth, were spawned together with the host oocytes. In most cases, a few donor progeny could be found among the offspring produced by the hosts. Donor progeny were examined with respect to an or gene-dependent maternal effect which normally causes wild-type eye color in homozygous ($\text{or}\text{or}$) larvae originating from the crossings of heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) females and homozygous ($\text{or}\text{or}$) males. This maternal effect was absent from homozygous ($\text{or}\text{or}$) larvae derived from homozygous ($\text{or}\text{or}$) donor oocytes which had developed in heterozygous females. Conversely, this maternal effect was observed in homozygous ($\text{or}\text{or}$) larvae derived from heterozygous ($\text{or}{}^{\mathrm{+}}\text{or}$) donor oocytes which had developed in homozygous ($\text{or}\text{or}$) host females. It is concluded that the oocyte genome is active at the or⁺ locus during oogenesis and that the oocyte is autonomous with respect to the product of synthesis of the or⁺ locus. In the present case, the “maternal effect” is therefore caused by synthetic activity in the growing oocyte. The results are discussed with respect to current information on gene products from animal genomes.     

Phosphorylated cardiac myofibrils and their effect on ATPase activity.

Control guinea pig cardiac myofibrils were isolated in the presence of Triton X-100. Experimental myofibrils, prepared in the presence of Triton X-100, NaF, cyclic AMP and ATP, possessed a reduced myofibrillar ATPase activity. When myofibrils isolated under control conditions were incubated for two hours at 25°C with NaF, ATP and cyclic AMP, the ATPase activity was also decreased; however, the ATPase activity was not reduced as much as that of myofibrils isolated under experimental conditions. Incubation of myofibrils with $\text{E. coli}$ aklaline phosphatase and guinea pig heart phosphoprotein phosphatase resulted in an increase in ATPase activity and a decrease in phosphoprotein phosphate. Thus there appeared to be an inverse relationship between myofibrillar ATPase activity and phosphoprotein phosphate content. The results indicated that a protein kinase is associated with the Triton X-100 purified myofibrils and supports the notion that intact myofibrils can exist in at least two catalytic forms.     

Induction and inhibition of meiotic maturation of follicle-enclosed porcine oocytes in vitro

Isolated porcine Graafian follicles which were explanted in vitro and maintained in organ culture were used as a test-system for the meiosis-inducing action of PMSG and hCG. The addition of either PMSG or hCG alone (10 or 20 IU/ml, respectively) to the culture medium was not effective, whereas the simultaneous administration of these hormones ($\text{15}\text{15}\text{IU/ml}$) induced resumption of meiosis in 90.3% ($\text{37}\text{41}$). The same hormone concentrations were used in a second series of experiments in which the inhibition and induction of meiosis of isolated oocytes were tested by transferring them into host follicles. In host follicles containing up to 12 foreign eggs, which were cultured in control media, meiosis was prevented in 86.0% of all oocytes ($\text{104}\text{121}$). By adding PMSG (15 IU/ml) simultaneously with hCG (15 IU/ml) to the medium, meiosis was induced in 95.0% of all oocytes ($\text{133}\text{140}$).The assumption is made that the signal initiating resumption of meiosis of the isolated and transferred oocytes is mediated by the follicular fluid, since intimate contact with the membrana granulosa of the host follicle was prevented by using a roller technique.     

Glucose repression of the inducible catabolic pathway for N-acetylglucosamine in yeast.

In order to investigate the mechanism of glucose repression of the N-acetylglucosamine metabolic enzymes in $\text{Candida}\text{albicans}$, an obligatory aerobic yeast, the activities of the following inducible enzymes were assayed: the N-acetylglucosamine uptake, N-acetylglucosamine kinase and glucosamine-6-phosphate deaminase. In the presence of glucose or other sugars e.g. succinate and glycerol, synthesis of these enzymes took place at a normal rate, suggesting that the hexose produces no catabolite repression in this organism. On the contrary, strong inhibition by glucose was observed on the activities of N-acetylglucosamine uptake and deaminase in N-acetylglucosamine-grown cells of $\text{Saccharomyces}\text{cerevisiae}$, a facultative aerobe. From the results, it is concluded that “glucose effect” or catabolite repression is absent in $\text{Candida}\text{albicans}$, a pathogenic strain of yeast.     

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [γ-³²P]ATP. The ³²P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and alkaline phosphatase. Two Ca²⁺-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La³⁺-ions (20 μM) in a similar way as the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na⁺. This phosphoprotein comigrates with a preparation of renal $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. In brush border membrane preparations the Ca²⁺-induced and the Na⁺-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca²⁺-pump and Na⁺-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg²⁺, the plasma membrane preparation exhibits a Ca²⁺-dependent ATPase activity of 2 μmol P_i per h per mg protein. It is suggested that this $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity is related to the measured Ca²⁺ transport which was characterized by $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for ATP and Ca²⁺ of $\text{44 ± 9 μ}\text{M}$ and $\text{0.25 ± 0.10 μ}\text{M}$, respectively. Phosphorylation of plasma membranes with [γ-³²P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca²⁺-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and from the catalytic subunit of $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca²⁺-pump in plasma membranes isolated from nucleated cells.     

The effect of growth rate,size, and season on oocyte development and maturity of Atlantic cod (Gadus morhua L.)

The effect of growth rate, body weight, age, and season on ovarian development and maturation was investigated for Atlantic cod (Gadus morhua L.) reared in the laboratory over 10 months, for each of two consecutive years, 1978–1980. Cod were also collected from the Gulf of St. Lawrence, the Scotian Shelf, Georges Bank, the Flemish Cap, and the N.E. Newfoundland Shelf.The state of maturity was recognized by oocyte size. Stage I oocytes did not vary in size with growth rate, season, age, or maturity, while the size of stage II oocytes was positively correlated with maturity and negatively correlated with maximum stage of development achieved. Ovarian wall thickness was positively correlated with age and maturity.The frequency distribution of stage I and II oocytes distinguished the state of maturation, with cod that would mature by the next spawning season having a minimum of 20% ($\text{x}\text{?}\text{= 42\%}$) of their oocytes at stage II or greater level of development.Maturing 3-yr-old cod had greater life specific growth rates than immature 3-yr-olds, but growth rates during the third year itself were not significantly different. A hypothesis of a three-part density-dependent mechanism controlling fecundity is postulated. Future reductions in partial recruitment and total fecundity are predicted for the Gulf of St. Lawrence cod stock based on calculated growth rates for Gulf cod in 1979.     

Changes in adenine nucleotide levels and respiration during 1-methyladenine induced maturation of starfish oocytes

The effects of 1-methyladenine on oxygen consumption and adenine nucleotide levels were examined in oocytes of Pisaster ochraceus and Patiria miniata. Oocytes of both genera to which 1-methyladenine was added consumed more oxygen than control oocytes beginning 1 to $\text{1}\text{1}\text{2}\text{h}$ after 1-methyladenine addition. The increase in oxygen consumption was correlated with maturation changes in the oocytes and particularly with germinal vesicle breakdown. Pre-fertilization oxygen consumption of eggs did not differ significantly from post-fertilization oxygen consumption of eggs in either genus for $\text{2}\text{1}\text{2}\text{h}$ after fertilization. ATP and AMP concentrations within the oocytes decreased during 1-methyladenine induced maturation, while the ADP concentration increased. It was suggested that increases in ADP concentration and decreases in ATP concentration within maturing starfish oocytes occurred in response to greater energy demands. The simultaneous increase in oocyte oxygen consumption was interpreted as an indicator of increased oxidative phosphorylation acting to restore initial nucleotide concentrations.     

Endogenous, cyclic 3'-5' AMP-dependent phosphorylation of human red cell pyruvate kinase.

Ribosomal proteins of $\text{Physarum}\text{polycephalum}$ were labelled $\text{in}\text{vivo}$ with ³²PO₄. Three acid phosphoproteins were observed in the large subunit, while two basic ones were present in the small subunit. Ribosomal phosphoprotein S3 accounted for 70% of the total radioactivity and may be equivalent to S6 from rat liver.     

The duplication of arginine catabolism and the meaning of the two ornithine carbamoyltransferases in Bacillus licheniformis.

Arginine deiminase and carbamate kinase activities are shown to be present in cell-free extracts of $\text{Bacillus licheniformis}$. This gives the rationale for the occurrence of an arginine-inducible ornithine carbamoyltransferase in this organism and suggests that, $\text{in vivo}$ and under the proper conditions, this enzyme is able to catalyze the phosphorolysis of citrulline. This also shows that this Bacillus species has two arginine catabolic pathways. This duplication appears to be under the control of O₂.