Effects of proteolytic and thermal action on the activity of lactate dehydrogenase isozymes LD-1 and LD-5.

L-Lactate dehydrogenase (LD) catalyzes the interconversion of pyruvate and lactate. Using a spectrophotometric assay to determine LD activity, incubation of rabbit, porcine, and bovine LD-1 and LD-5 isozymes with the protease subtilisin (Carlsberg) gave first-order degradation kinetics. Degradation half-lives were significantly lower for the LD-5 isozymes from the three species when incubated with subtilisin at temperatures from 4 degrees C to 25 degrees C. The energy involved in the degradation process, however, was not different. The activation energy for the conversion of pyruvate to lactate by LD-1 at pH 7.4 was significantly higher than that for LD-5 for all three species examined (P less than 0.005). Thermocalorimetry showed that the LD-1 isozymes have both a higher mean temperature of denaturation and a higher heat uptake during the denaturation process than corresponding LD-5 forms. The results suggest that the LD-5 isozymes in the species studied are more metabolically efficient, whereas the LD-1 forms have greater structural stability.

Lactate dehydrogenase in rat mitochondria.

Small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from rat heart, kidney, liver, and lymphocytes. Brain mitochondrial preparations were also isolated, but the results were inconclusive. A variety of cytosolic markers were used and it was found that essentially no cytosolic contamination was present except in brain preparations. A bacterial protease was used along with digitonin fractionation to determine localization of the mitochondrial LDH. Approximately 80% of the LDH activity associated with heart and kidney mitochondrial preparations was on the inside compared to about 40% for liver. Lymphocyte mitochondrial LDH activity was about 70% on the inside. Cytosolic LDH-5 preferentially adheres to outer mitochondrial membrane of liver, kidney, and heart. Agarose gel electrophoresis showed LDH isozymes in mitochondria qualitatively similar to that of the corresponding cytosol except in kidney mitochondrial preparations, where a specific electrophoretic band was found which did not correspond to any of the common LDH isozymes.

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity.

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.

Localization of L-lactate dehydrogenase in mitochondria.

Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.

Evidence for polyphosphate in phosphorylated nonhistone nuclear proteins.

Structural studies of eucaryotic nuclear proteins have revealed the presence of bound polymeric phosphates. 32P-labeled and nonlabeled nonhistone nuclear proteins (NHPs) were isolated from rat liver nuclei and subjected to various controlled hydrolytic conditions. The analysis of protease-trypsin limit peptides revealed the presence of six phosphorylated, homogeneous fragments with phosphate/amino acid molar ratios greater than unity, ranging from 1.3 to 79. Alkaline beta elimination of phosphoester bonds released polymeric phosphates with chain lengths from 2 to over 200, as determined by using two-dimensional chromatographic analysis. The identity of these labeled polymeric phosphates was established to be polyphosphate by a number of criteria, including chromatographic mobility, gravimetric precipitation to constant specific activity, generation of orthophosphate on hydrolysis, and the determination of the delta H of hydrolysis of phosphoanhydride bonds. The evidence suggests that, in addition to the phosphomonoesters of serine and threonine, multiple phosphoanhydride linkages can result in the formation of polyphosphorylated NHPs. Previous investigators have demonstrated that exogenous, free polyphosphate causes destabilization of chromatin and enhancement of transcription in vitro. Although the function of the polyphosphorylated NHPs is currently unknown, such findings have possible functional implications with regard to the postulated role of NHPs as positive modifiers of gene expression.

D- and L-lactate catabolism to CO2 in rat tissues.

The current study was initiated in order to compare the rates of oxidative catabolism of D- and L-lactate in various rat tissues. Uniformly labeled D- or L-[14C]lactate was incubated at 37 degrees C in a closed system with tissue homogenates in Krebs-Ringer phosphate buffer. Evolved 14CO2 was trapped in a center well containing a fluted filter paper saturated with strong base and the radioactivity determined. The ratio of L-lactate to D-lactate oxidation was greatest in brain, followed by kidney, heart, and liver. In liver the rate of oxidation of D-lactate exceeded that of L-lactate, in heart the rates were not significantly different and in the other two tissues L-lactate was oxidized more rapidly than D-lactate. These results indicate that the rate of D-lactate catabolism is considerable and is relatively greater than had been reported previously.

A model system for direct in vivo measurement of absorbed nutrients in portal venous blood. Applicability to alcohol studies.

In order to investigate the in vivo effect of alcohol on intestinal absorption and on other factors which influence the rate of appearance of nutrients into the portal venous system, an experimental system has been developed in which separate cannulas have been implanted in the duodenum and portal vein of the rat. Following recovery from surgical procedures, unanesthetized animals may be administered nutrients directly into the duodenum, with and without alcohol, and the rate of appearance of nutrients in the portal system may be monitored by rapid sampling of the portal blood. Potential applicability of this experimental animal is demonstrated by the kinetics obtained with an amino acid, L-phenylalanine, both in the presence and the absence of alcohol.