Characterization of a C-kinase inhibitory fraction

A broad area of C-kinase inhibitory activity was detected after DEAE-cellulose chromatography of 20,000 g supernatant of rat stomach homogenate. Attempts to characterize the inhibitory moiety indicated that it was a non-protein, low molecular weight substance which eluted immediately after C-kinase activity. Similar inhibition was observed in the presence of KC1, the salt used in the elution buffer. Several other salts examined also displayed inhibition of C-kinase activity. Kinetic analysis showed that both KC1 and the inhibitor displayed competitive inhibition of histones and gave identical inhibitor constants.     

Multiple fraction collection using a peristaltic pump and a fraction collector modified for multiple channel operation

The fractionation of gradients in sedimentation analysis of proteins is a time-consuming operation. This operation can be performed more rapidly by using a multichannel peristaltic pump and two or more fraction collectors. Specifically designed fraction collectors are available for multiple fraction collection, notably the Slave Micro-Fractionator (Model SFC-80)¹ from Gilson Medical Electronics, Inc. Use of multiple fraction collectors during the fractionation of gradients has the disadvantage that these units occupy considerable space and are expensive. In addition, the total number of fractions collected from one gradient is often considerably less than the capacity of most fraction collectors. To offset the space and expense disadvantages, we have devised a modification for the Gilson Micro-Fractionator (Model FC-80H)¹ which permits three sets of 25 fractions each to be collected simultaneously from 3 gradients using only 1 fraction collector. A multichannel peristaltic pump is employed, and an attachment which permits three drop tubes to be positioned above the rack of fraction tubes is secured to the drop detector head¹ of the Micro-Fractionator. One drop tube is clamped in the normal manner in the drop detector head, and fraction size is determined by counting drops in the normal manner. Fractions from the other two drop tubes are not counted. Alternatively, fraction size can be determined by adjusting the pump speed and using the timer on the fraction collector.     

Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of γ-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in γ-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in γ-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed γ-glutamyl transpeptidase activity localized in the capillary walls.     

Characterization of a heat-stable fraction of human IgG

Heat aggregation of human IgG has been studied by photon correlation spectroscopy, ultracentrifugation, circular dichroism, and differential scanning calorimetry. It is found that pooled human IgG can be separated into two fractions of molecules, one that easily aggregates and one that is stable upon heating. In a buffer atpH=7.6 and 0.2 M NaCl it is found that about half of the original monomeric molecules do not aggregate even after heating at 62°C for 24 h. No differences in the antigen binding capacity of the heat-stable fraction and normal IgG are observed. Heat-stable molecules can partially be transformed to heat-aggregating molecules by a rapid acid denaturation followed by neutralization. Differential scanning calorimetry shows that the major heat denaturation, which is a two-phase process atpH=7.6, starts at about 63°C. Only minor differences between the heat-stable and the heat-labile fractions are observed in the thermograms. No differences are observed in the far-UV region of the CD spectra, indicating that the secondary structure of the heat-stable IgG does not differ from the native IgG molecule. While the aggregation of normal human IgG can be described by Smoluchowski kinetics, the heat-stable fraction follows another kinetics, which includes an activation step.     

Polymorphism in a casein fraction of sows' milk

Polyacrylamide vertical gel electrophoresis was used to separate the caseins of sows' milk. Polymorphism was found in a region of the gels designated Cn₃. The Cn₃ polymorphism consists of two bands which appear to be controlled by two codominant alleles designated Cn ₃ ^A and Cn ₃ ^B . Homozygotes possess one band and heterozygotes posses both bands of equal intensity giving the following phenotypes: Cn₃A, Cn₃AB, and Cn₃B. A chi-square test for goodness of fit of the observed phenotypes to that expected by the Hardy-Weinberg equilibrium formula and a segregation analysis of eight matings involving 19 female progeny conform to the hypothesis that the Cn₃ polymorphism is controlled by two codominant autosomal alleles. Further family studies will be necessary to confirm the genetic control of the Cn₃ polymorphism.Deceased.     

Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus.

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of gamma-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in the gamma-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in gamma-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed gamma-glutamyl transpeptidase activity localized in the capillary walls.     

An enzymic analysis of a nuclear envelope fraction

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104–125 (1972)) is characterized by high levels of glucose-6-phosphatase and 5′-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes $\text{b}{}_5$ and $\text{P-450}$ as well as NADPH- and NADH-cytochrome $\text{c}$ reductase activities are present but at lower levels than found in microsomes. Cytochrome $\text{c}$ oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5′-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside trisphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.