The reactions of pyridoxal 5′-phosphate with the M4 and H4 isoenzymes of pig lactate dehydrogenase

The H₄ and M₄ isoenzymes of pig lactate dehydrogenase are both inactivated by reaction with pyridoxal 5′-phosphate. In the early stages, inactivation is largely reversible by the addition of lysine in excess, but may be made irreversible by reduction with borohydride. This indicates that modification of lysine residues probably causes the initial inactivation. Both isoenzymes also undergo a slower process of irreversible inactivation which becomes more evident with increasing concentrations of pyridoxal 5′-phosphate and higher temperature. Although coenzymes give only partial protection of enzyme activity, they nevertheless completely prevent irreversible inactivation. Neither pyruvate nor lactate alone gives any protection. With the M₄ isoenzyme, complete protection against inactivation by pyridoxal 5′-phosphate may be achieved in ternary complexes, but no conditions have been found for complete protection of the H₄ isoenzyme. In the course of irreversible inactivation of H₄ lactate dehydrogenase, complete loss of activity can be correlated with the loss of approximately two free thiol groups per subunit. Present findings with regard to the importance of temperature and reagent concentration in determining the outcome of the chemical modification appear to resolve earlier controversy.     

Polyadnylic acid sequences in RNA able to transfer specific delayed type hypersensitivity in vitro.

Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with 10^?3M cycloheximide, (Cy)], although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.     

Anomalous behavior of certain poliovirus polypeptides during SDS-gel electrophoresis.

Urea is shown to be a useful additive to sodium dodecyl sulfate gels prior to electrophoresis and offers an alternative means for the resolution of some SDS-protein complexes. Two types of effect are described, one of which causes increases in the relative mobilities of certain polypeptides found in poliovirus-infected cells. It is postulated that urea plus SDS is able to achieve a more complete denaturation of some polypeptides, which leads to faster electrophoretic mobilities. The molecular weight estimations for such proteins in these conditions are therefore lower than those determined in the presence of SDS alone. A second effect of some urea solutions is to cause the multiple banding of the structural polypeptide VP3 when included in gels at high concentrations. This effect is variable and appears to be unrelated to the presence of cyanate ions.     

Serum-independent human lymphoblastoid cells. Effects of cell density on growth

A serum-independent (SI) line of human lymphoblastoid cells has been developed from a clone of the serum-dependent (SD) line RPMI 8866. The SI cells have been growing in serum-free culture for more than one year. In high serum concentrations the growth of the SI cells is identical to that of the SD cells. At low serum concentrations the SD cells die while the SI cells survive and grow. The growth of SI cells is density-dependent and can be overcome by the addition of serum, conditioned medium, or daily feeding with fresh medium.     

Photoreactivating enzyme from Euglena and the control of its intracellular level

Photoreactivating (PR) enzyme activity has already been demonstrated by us in cell-free extracts of Euglena gracilis var. bacillaris Pringsheim using the Hemophilus transformation assay. This activity can also be detected in extracts using a direct non-biological assay for the photorepair of thymine dimers in DNA. PR enzyme is found in extracts of both wild-type cells and cells of an aplastidic mutant, W₃BUL, lacking detectable chloroplast DNA, indicating that the PR enzyme is neither coded nor translated exclusively in the chloroplast, but is probably coded in the nucleus and translated in the cytoplasm. Growing cultures of wild-type cells manifest a large increase in PR enzyme activity in vitro upon entering stationary phase. This correlates with the increased photoreactivability of chloroplast inheritance in vivo in stationary phase cells, previously found for Euglena, and suggests that a substantial part of the newly synthesized PR enzyme is available to repair plastid DNA. When dark-grown nondividing wild-type cells are exposed to light, there is a large increase in the specific activity of PR enzyme measured in vitro. This increase is prevented by cycloheximide but not by chloramphenicol or streptomycin, indicating that the enzyme is synthesized on 87s cytoplasmic ribosomes rather than 68s chloroplast ribosomes. Wavelengths of light effective for PR of chloroplast DNA in vivo are also effective for the light induction of PR enzyme. A brief illumination (45 min) of dark-grown nondividing wild-type cells triggers the synthesis of PR enzyme which continues in the absence of light. Growing cultures of W₃BUL also exhibit a preferential synthesis of PR enzyme in the staionary phase of growth, but the specific activity in vitro is consistently ten times higher than that of wild-type. Dark-grown non-dividing cultures of W₃BUL also show a cycloheximide-sensitive light induction of PR enzyme synthesis which, however, is dependent on the continued presence of light. The light induction of PR enzyme synthesis can be regarded as the induction of an enzyme by one of its substrates.     

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide at the peptide bond. l-Phenylalanyl-l-leticine amide, N-carbobenzoxy-l-leucyl-l-phenylalanine amide, N-carbobenzoxy-l-leucyl-l-pheml-alanine, N-carbobenzoxy-l-phenylalanyl-l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, vizl-leucyl-l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl-l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl-l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.     

A major role for chloride in (3H)- noradrenaline transport by rat heart adrenergic nerves

The effect of Cl^? and other anions on (³H)-noradrenaline line (NA) transport by bisected rat heart atrial appendages $\text{in}$$\text{vitro}$ has been studied. It was found that NA active transport, at the plasma membrane level, shows an absolute dependency on Cl^?, with a half-maximal activation of transport occurring at 6 mM Cl^? and complete saturation at 50 mM. Cl^? effects are due to changes in transport Km, while Vmax is not changed. Only one class of sites for Cl^? seem to be present in the transport system. Br^? can substitute for Cl^? with 90% effectiveness, nitrate and iodide are less effective, while larger anions are very poor substitutes. In addition, heart atrial hemi-appendages have been characterized as a suitable preparation for studies of this type.     

Separation of bases, ribonucleosides and deoxyribonucleosides by anion-exclusion and partition chromatography on cation-exchange resin: application to the assay of ribonucleotide reductase, deaminase and nucleosidase.

[5-³H]CDP and CTP are used as substrates in the assay of ribonucleotide reductase, deaminase and nucleosidase activity in crude enzyme preparations. After incubation, the nucleotides are hydrolyzed to nucleosides by sequential treatment with potato apyrase and alkaline phosphatase. An aliquot is then chromatographed on a cation-exchange column at 50°C with 0.1 m boric acid, adjusted to pH 7.4 with ammonia, used as eluant. The pyrimidines Ura, Urd, dUrd, Cyt, Cyd and dCyd are separated and eluted in about 50 min in small volumes. Assays by this procedure of CTP reductase activity in crude fractions of ribonucleotide reductase from Euglena gracilis gave results comparable to those obtained by the standard method. The new procedure is also applicable when adenine or guanine nucleotides are used as substrates. The adenine derivatives Ade, Ado, dAdo, Hyp, Ino, dIno as well as the guanine derivatives Gua, Guo, dGuo, Xan, Xao are separated from each other in this chromatographic system in about an hour.     

A method for determining amino acid concentrations and specific activities of amino acids and some other compounds in biological fluids

A method is described for obtaining plasma ultrafiltrates from which the concentrations of all amino acids, including tryptophan and ammonia, are obtained. A split-stream methodology is described for obtaining, in addition to the concentrations, the radioactivities of amino acids, glucose, and plasma water.     

Effects of γ-aminobutyric acid on 33Pi incorporation into rat brain phospholipids in vivo

The effects of γ-aminobutyric acid (GABA), bicuculline and strychnine on the incorporation $\text{in vivo}$ of ³³Pi into phospholipids of rat brain were studied at 10 and 30 minutes after intracisternal injection of the radionuclide. GABA inhibited labeling of phospholipids in the three brain regions studied at both times. Bicuculline by itself had no significant effect on ³³Pi incorporation, but totally blocked the inhibitory effect of GABA in all three brain regions. Strychnine by itself inhibited phospholipid labeling in the brain stem and forebrain, had no significant effect on GABA inhibition of ³³Pi incorporation in the cerebellum and forebrain, and partially blocked the GABA effect in the brain stem. GABA inhibited ³³Pi incorporation into phosphatidic acid, phosphatidylinositol, phosphatidyl choline and phosphatidyl ethanolamine but had no effect on phosphatidyl serine. The data suggest that the inhibitory effects of GABA on CNS phospholipid labeling are mediated specifically through GABA receptor sites.     

A simple, versatile, nondisruptive method for the isolation of morphologically and chemicaly pure basement membranes from several tissues.

A simple procedure has been developed for the isolation of ultrastructurally pure, intact basement membranes from bovine retinal and brain blood vessels, rabbit renal tubules and rat renal glomeruli. By this procedure, cell membranes and intracellular materials are selectively solubilized with 4% sodium deoxycholate to yield morphologically and chemically intact basement membrane preparations. Therefore, this method appears to be a versatile, nondisruptive procedure for the isolation and characterization of basement membranes from a variety of tissues. Its applicability has been demonstrated by the preparation for the first time of isolated basement membranes from non-renal mammalian blood vessels.     

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10⁶-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp. Physiol. Ser. B130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physiol. Biochem.87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. (Arch. Biochem. Biophys.196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.     

Morphological observations on cultured lampbrush-stage Rana pipiens oocytes.

Early lampbrush-stage oocytes are characterized by small lampbrush chromosome loops, a small amount of ribonucleoprotein (RNP) matrix on the loops, small nucleoli, few RNP particles in the nucleoplasm, and a smooth germinal vesicle contour. In vitro culture of these oocytes in serum-free culture medium for 24 hr at 18°C promotes a number of morphological changes in the oocytes: The lampbrush loops increase in diameter and acquire extensive RNP matrix, the nucleoli increase in size and complexity, the nucleoplasm accumulates numerous polymorphic RNP particles, and the germinal vesicle envelope acquires a sacculated contour. These characteristics are typical of the in vivo maximum lampbrush stage, and their appearance is due to an apparent in vitro acceleration of the lampbrush phase. Two possible interpretations of these observations are discussed.     

Relationship between growth and meiotic maturation of the mouse oocyte

Oocytes of various sizes were isolated from trypsinized ovaries of juvenile mice, cultured in a chemically defined medium, and scored for the resumption and completion of meiotic maturation. Oocytes recovered from mice younger than 15 days remained in the germinal vesicle stage, whereas those from mice 15 days or older resumed meiosis at a frequency which increased with the age of the mice. The mean diameter of the oocytes recovered also increased with the age of the mice. Within individual litters, the mean diameter of oocytes which failed to mature (incompetent oocytes) was significantly less than that of oocytes which matured (competent oocytes). The frequency of premature metaphase I arrest decreased markedly as the age of the mice and oocyte volume increased. These results suggest that the ability to resume meiosis is acquired at a specific stage of oocyte growth in the juvenile mouse, and that the ability to complete meiotic maturation is acquired subsequently. These oocytes provide an in vitro system with which to study the control of meiosis in the mammal.