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Biomedical subjects

M M Werber

Publications and source records attributed to M M Werber.

At least 37 records · Page 2Linked to original sources
PubMed1986

NADH-dependent prostaglandin E2-9-ketoreductase activity and prostaglandin synthesis in the Brattleboro rat kidney: effects of the antidiuretic hormone.

The activity of prostaglandin (PG)E2-9-ketoreductase (9KR), an enzyme catalyzing the conversion of PGE2 to PGF2 alpha, was significantly increased in glomerular and cortical homogenates of diabetes insipidus (DI) rats, as compared to normal Long Evans (LE) rats, and did not change with ADH treatment. Medullary 9KR was similar in the three groups and papillary 9KR was increased, but not significantly, in both groups of DI rats. Km values for PGE2 and NADH were compared in the various compartments of the kidney. Levels of 9KR were not correlated with the PGE/PGF ratio in urine or supernatants. The synthesis of PGE2 and PGF2 alpha by isolated glomeruli was increased in DI rats. This was not reversed by ADH treatment, PGE2 synthesis increasing even further, especially in the presence of arachidonic acid. In contrast, medullary slices produced significantly less PGs in DI than in LE rats and returned to normal with ADH treatment. Papillary slices produced similar quantities of prostaglandins in all groups. The results do not support the concept that the alterations in PG synthesis observed in DI rat are related only to changes in 9KR activity, but do not exclude the possibility that the enzyme participates in the regulation of PG biosynthesis.

Animals↗
PubMed1986

Effect of the integrity of the myofibrillar structure on the tryptic accessibility of a hinge region of the myosin rod.

Limited proteolysis has been used to study the influence of actin, in the absence or presence of regulatory proteins of the thin filament (tropomyosin and troponin), as well as that of the myofibrillar structure on the tryptic cleavage of the heavy meromyosin (HMM)/light meromyosin (LMM) hinge region in myosin heavy chain. Cleavage at the HMM/LMM hinge is almost absent in myofibrils, whereas this hinge is accessible to tryptic digestion in actomyosin, in native thin filaments attached to myosin and in myosin heavy chain alone. This observation indicates that it is the myofibrillar structure which profoundly affects the tryptic accessibility of this specific hinge region of myosin. This provides a good example of the manner by which a highly organized supramolecular structure might affect the chemical properties of a specific site in a macromolecule.

Actins↗
PubMed1985

Identification and purification of human T-lymphocyte colony-enhancing factor, TLCEF: increased production by phorbol myristate acetate.

T-lymphocyte colony-enhancing factor (TLCEF), a growth factor for a minute subpopulation of T lymphocytes, is produced, along with other factors, by conditioned media (CM) of mononuclear cells following stimulation with T mitogens, such as phytohaemagglutinin (PHA). A combination of PHA and a co-mitogen, phorbol 12-myristate 13-acetate (PMA), has been shown to have a synergistic effect on the production of TLCEF, yielding levels of activity eight to fifteen times higher than those obtained with either PHA or PMA alone. TLCEF was purified by ammonium sulphate, fractionation hydrophobic interaction chromatography on phenyl-Sepharose and gel filtration. The last step of this purification procedure yielded two peaks of activity purified 13,000- and 29,000-fold, respectively. The first peak eluted from the column with an average molecular weight of 125,000, whereas the second peak was retained on the gel, probably due to non-specific interactions with it. The purified fractions contained none of the following activities: T-cell growth factor (TCGF), colony-stimulating factor-1 (CSF-1), interleukin-3 (IL-3) and interferon. TCGF activity, which is known to be unstable at high degrees of purification, was already lost after the ammonium sulphate fractionation step, most probably because of the low protein and albumin concentrations (0.33 and 0.11 mg/ml, respectively). TLCEF is thus a more stable immunoregulatory factor than TCGF and has a much higher apparent molecular weight.

Chromatography, Gel↗
PubMed1984

Hydrophobic interaction chromatography of myosin fragments: potential use in purification.

Myosin fragments were fractionated on columns of the hydrophobic gel phenyl-Sepharose CL-4B. In the presence of high NaCl concentrations the fragments bound tightly to the columns; they could be eluted by decreasing the ionic strength, by increasing the pH, or by applying various concentrations of ethylene glycol. In myosin subfragment-1 (S-1), the light chains underwent partial dissociation from the heavy chain and bound separately to the column matrix. The order of strength of binding of the various species to the column was heavy chain greater than A1 light chain greater than A2 light chain greater than native S-1 greater than denatured heavy chain or S-1. Thus the hydrophobic gel appears to be able to differentiate between enzymatically active and inactive S-1. Under appropriate elution conditions it was possible to obtain S-1 preparations depleted from nicked heavy chains and with specific ATPase activities 34-130% higher than those of untreated S-1. When S-1 (A2) was fractionated on phenyl-Sepharose a fivefold enrichment of the heavy chain with respect to the light chains was obtained, while the ATPase activity was equal or larger than that of the original S-1, implying that the light chains are not essential for ATPase activity. Thus, it seems that chromatography of S-1 on phenyl-Sepharose is a potentially useful method for obtaining a purified myosin heavy-chain fragment with a high ATPase specific activity.

Adenosine Triphosphatases↗
PubMed1983

Identification and partial purification of human T lymphocyte colony-enhancing factor (LCEF), distinct from T cell-growth factor.

Colony growth of T lymphocytes in a soft agar culture system, following stimulation with phytohaemagglutinin (PHA), is enhanced by T cell-derived conditioned medium (CM). The putative enhancing factor present in this CM has been termed lymphocyte colony-enhancing factor (LCEF). The purification of LCEF from CM has been complicated by the fact that active CM could be obtained only in the presence of 7-8 mg/ml of serum proteins. In order to circumvent this problem, CM which was shown to be active in the colony formation assay, was produced in the presence of a serum fraction precipitated by 1.6 M ammonium sulphate (AS). The first step of the purification of this CM was fractionation by 1.6 M AS, whereby the active material was fully recovered in the supernatant. The 20-fold purified material was applied on a DEAE-cellulose column. Two peaks of activity were obtained using the colony formation assay. The purification achieved by this second step was 5-10 fold for the first peak, eluting around 0.06 M NaCl, and two- to three-fold for the second peak, eluting around 0.19 M NaCl. Two peaks of activity were also obtained upon hydrophobic chromatography on a phenyl-Sepharose column, with enhancement factors of 5-8 for the first peak and 25-60 for the second peak. These results imply the presence of more than one LCEF molecule in the original CM. None of the LCEF-active fractions contained any T cell-growth factor (TCGF) activity; LCEF seems therefore to be a molecular entity distinct from TCGF.

Chromatography, Gel↗
PubMed1982

Binding of calcium and magnesium to myosin in skeletal muscle myofibrils.

Binding profiles for divalent cation to myosin have been obtained in myofibrils where myosin is assembled in arrays typical of the in vivo organization. Protection by Ca2+ and Mg2+ ions of the regions of myosin susceptible to chymotryptic attack provided the means to monitor metal ion binding. The effect of various concentrations of divalent cations on the chymotryptic digestion patterns was assessed by densitometry of Coomassie Blue stained gels obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by the rate of myofibrillar solubilization. The results indicate the presence of two classes of binding sites differing in affinity by 4 orders of magnitude. The fractional saturation of the high-affinity site associated with the 5,5'-dithiobis(2-nitrobenzoic acid)-dissociable light chains of myosin regulated the production of subfragment 1 of myosin. From the digestion profiles as a function of metal ion concentration, binding constants for Mg2+ and Ca2+ were obtained. The value for Mg2+ was 5.7 x 10(6) M-1, which is about 1 order of magnitude higher than the most recently determined values for free myosin in solution; the value for Ca2+ was 6.3 x 10(6) M-1. Binding to the low-affinity site regulated the production of the heavy meromyosin fragment and yielded association constants for Ca2+ and Mg2+ of 0.9 x 10(3) and 0.7 x 10(3) M-1, respectively.

Animals↗
PubMed1982

Inhibition of the Ca2+-Mg2+ ATPase of sarcoplasmic reticulum by Co-(phen)-ATP.

Active Ca2+ transport in sarcoplasmic reticulum derived from skeletal muscle is shown to be inhibited by an ATP derivative Co(III)-phenanthroline-ATP. The inhibition is by competition for the MgATP binding site on the Ca2+-ATPase pump molecule. For preincubation times of 30 min or less, no quasicovalent bonds are established between the inhibitor and the ATPase. The apparent dissociation constant for the enzyme-inhibitor complex is 0.27 mM), a value close to the apparent Km and Ki values for MgATP (0.24 mM) and CoATP (0.14 mM). Addition of Co-(phen)-ATP to sarcoplasmic reticulum after maximal Ca2+ uptake has been achieved causes the release of the accumulated Ca2+. Under the experimental conditions chosen, the release occurs in two phases: one with a half-time of less than 2 sec and one with a half-time of 17 to 50 sec. The implications of these findings to the ATPase active transport mechanism are discussed.

Adenosine Triphosphate↗
PubMed1982

Actin, myosin and actin-binding protein in lymphoid cells from human normal and leukemic cell lines.

Actin, myosin and actin-binding protein have been identified in lymphoid cell lines of Null, B or T type of normal and malignant origin. The cells were fractionated into cytosol (Fraction 1), high KC1 extract (Fraction 2) and mainly membrane (Fraction 3). The distribution of the three contractile proteins and the ATPase activities of myosin and actomyosin in B lymphoblastoid normal and B-lymphoma malignant cell lines. Actomyosin-ATPase activity in the normal B line was much higher than it was in the Null B or T malignant lines examined. These results agree with the assumption that malignant transformation is accompanied by an impairment of the action of contractile proteins that leads to a decrease in motility.

Actins↗
PubMed1980

Amino acid sequence of 2Fe-2S ferredoxin from an extreme halophile, Halobacterium of the Dead Sea.

The primary structure of the 2Fe-2S ferredoxin from Halobacterium of the Dead Sea was determined and it consisted of 128 amino acid residues including an N epsilon-acetyllysyl residue. Due to a high degree of sequence homology between this ferredoxin and the one from Halobacterium halobium, all tryptic peptides could be aligned in order. Only 20 amino acid differences were observed between these two halobacterial ferredoxins. The distribution of cysteinyl residues involved in the iron chelation was similar to that of chloroplast-type ferredoxins.

Amino Acid Sequence↗
PubMed1980

[Halophilism].

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Adaptation, Physiological↗
PubMed1979

Viscosity-dependent structural fluctuations in enzyme catalysis.

The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.

Carboxypeptidases↗