James F. Lenney

Activities and cellular localization of yeast proteases and their inhibitors

We have found that proteases A, B and C of Saccharomyces cerevisiae are localized in the vacuole. The corresponding inhibitors of these three proteases are present in the extravacuolar cytosol. The compartmentation of the yeast proteases suggests that their function is primarily the intravacuolar digestion of proteins. Yearst cells were grown under various conlitions, and it was found that culture conditions which either induce biochemical differentiation of cells or which do not allow growth result in the enhancement of proteolytic activities.

Purification and properties of human serum carnosinase

Carnosinase from human plasma was purified 18,000-fold to apparent homogeneity in a four step procedure. The dipeptidase was partially inactivated during DEAE-cellulose chromatography; however, it reactivated slowly when concentrated and stored at 4 degrees C. In the second purification step, hydroxylapatite column chromatography, two forms of the enzyme were separated from one another. Human serum carnosinase was found to be a glycoprotein with a pI of 4.4 and a subunit Mr of 75,000; the active enzyme was a dimer, the two subunits being connected by one or more disulfide bonds. The enzyme was especially active in hydrolyzing carnosine and anserine, preferring dipeptides with histidine in the C-terminal position. In most human tissues, the concentration of serum carnosinase was proportional to the percentage of trapped blood in the sample. However, the brain contained about 9 times more enzyme than expected, based on the amount of trapped blood present. The physiological function of this enzyme seems to be the hydrolysis of homocarnosine in the brain and the splitting of carnosine and anserine in the blood stream. Six higher primates were found to have serum carnosinase. Twelve nonprimate mammals were tested; all were lacking the serum enzyme except for the Golden hamster, which had very high concentrations of a carnosinase having somewhat different properties than the higher primate enzyme.

Human serum carnosinase: characterization, distinction from cellular carnosinase, and activation by cadmium.

Human serum carnosinase was assayed using a simple and sensitive fluorometric method. Under optimum conditions, the average adult serum hydrolyzed 42 mu mol of carnosine per ml per hour, about 17 times the average activity reported in the literature. Cadmium was twice as effective as manganese as an activator of this enzyme. Serum carnosinase was found to be different in many respects from cellular carnosinase. For example, the serum isozyme hydrolyzed homocarnosine, whereas the cellular carnosinase did not. The apparent molecular weight of serum carnosinase was 160 000, while that of the cellular isozyme was 90 000. Although it has been reported that serum contains two molecular forms of carnosinase, only one form was detected using several electrophoretic methods and two ion exchange chromatography procedures. The concentration of serum carnosinase varied greatly between individuals. Little or no enzyme was detected in children below 10 months in age. Thereafter, the average concentration of carnosinase increased gradually to reach the adult range at age 13-15.

Purification and properties of two proteinases from Saccharomyces cerevisiae

Abstract A procedure is described for the purification of two intracellular proteinases from bakers yeast (Saccharomyces cerevisiae). These enzymes (which were not separated by gel filtration or ion-exchange chromatography) are either very similar or form a complex with one another. After the final purification step, each proteinase had a specific activity 25% greater than that of crystalline trypsin. The proteinases have isoelectric points below pH 4.8, and molecular weights of 73,000 as measured by Sephadex G-100 chromatography. Crude preparations, and a few of the purified fractions, showed good stability at 4 °. Most ion-exchange chromatography preparations gave low enzyme yields, possibly because of the loss of a stabilizing factor or the removal of a third proteinase. Metal chelators and alkaline earth metals had no effect on activity or stability, suggesting that these proteinases are not metallo-enzymes. Proteinase B is inhibited by p-chloromercuribenzoate (PCMB) and mercury ions; PCMB inhibition is partially reversed by cysteine, while mercury inhibition is fully reversed. Eighty per cent or more of each proteinase in the yeast cell is in the zymogen form and seems to be particle-bound.

Specificity and distribution of mammalian carnosinase

Hog kidney carnosinase (EC 3.4.13.3) was found to have a narrow specificity; it hydrolyzed carnosine, anserine and glycyl-L-histidine, but did not split L-alanyl-L-histidine or homocarnosine. The isoelectric point of this enzyme was 5.8 and its molecular weight was about 84 000. Carnosinase was found to be widely distributed in various tissues of the rat. Uterus, kidney, liver and lung contained high levels of carnosinase, whereas moderate concentrations were found in spleen, heart and brain, with low levels in small intestine, skeletal muscle and stomach, and none in blood.

Homocamosinosis: lack of serum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine

Patients afflicted with homocarnosinosis have elevated concentrations of homocarnosine in brain and CSF. It has been reported that they lack brain homocarnosinase. However, we have found that these patients are deficient in serum carnosinase, a dipeptidase which hydrolyzes homocarnosine about 5% as rapidly as it splits carnosine. Homocarnosinase could not be detected in normal human brain extracts after isoelectric focusing or DEAE-cellulose column chromatography. The ability of brain extracts to hydrolyze homocarnosine thus appears to be attributable solely to the serum carnosinase which is present because of serum trapped in the brain sample. Preliminary data indicate that homocarnosinase is probably not present in 13 other human tissues. Normal CSF contained serum carnosinase, whereas the CSF of a homocarnosinosis patient was lacking this enzyme. Thus it appears that the elevated concentrations of homocarnosine in the CSF of homocarnosinosis patients are attributable to serum carnosinase deficiency.

Localization of a novel pathway for the liberation of GABA in the human CNS

Serum carnosinase is a dipeptidase, which is synthesized in human brain, where it hydrolyzes homocarnosine to release free GABA. Immunohistochemical procedures were used to demonstrate the presence of this enzyme in several layers of the retina and in certain neuronal tracts of the cerebral cortex, cerebellar cortex, olfactory bulb, hippocampus, and in disseminated tracts presumably from the internal capsule, interspersed among the basal ganglia. The enzyme was also present in the epithelial cells of the choroid plexus and in corpora amylacea, which were seen in many regions of the CNS. Homocarnosine was localized either in the same tracts or in nearby neurons. For example, the Purkinje cells of the cerebellar cortex contained homocarnosine, whereas serum carnosinase was localized in adjacent neuronal projections apparently originating from outside the cerebellar cortex and having probable synaptic contact with the Purkinje cells. These findings suggest that in addition to glutamate decarboxylation, a second metabolic reaction for the formation of free GABA exists in specific neuronal tracts of the human CNS where GABA is released from homocarnosine by the action of serum carnosinase.

LOW MOLECULAR WEIGHT INHIBITORS OF CATHEPSINS B, H AND T IN HUMAN SERUM, SYNOVIAL FLUID AND CSF

Summary When human plasma was chromatographed on a Sephadex G-75 column, cathepsin B inhibitors with molecular weights of 80,000 and 13,000 were eluted. The high molecular weight peak (TPI-H) contained α-thiol proteinase inhibitor and haptoglobin, both of which inhibited cathepsins B and H. The low molecular weight peak (TPI-L) inhibited cathepsins B, H and T, but had little or no activity against papain, ficin or bromelain. TPI-L from plasma contained two molecular forms of inhibitor, while TPI-L from pooled serum samples contained nine. Serum TPI-L was found to be different from the low molecular weight inhibitors present in lung and many other organs. When rheumatoid synovial fluid was analyzed by gel filtration, the eluate contained TPI-H and TPI-L. Human cerebrospinal fluid eluates contained TPI-L, but not TPI-H.

Homocarnosinase: A hog kidney dipeptidase with a broader specificity than carnosinase

Abstract A dipeptidase was isolated from hog kidney; it is the first enzyme described that has the capacity to cleave homocarnosine. It was purified to apparent homogeneity and split carnosine, anserine, and several other dipeptides in addition to homocarnosine. Homocarnosinase had a molecular weight of 57,000 as determined by sodium dodecyl sulfate-gel electrophoresis; it appeared to consist of a single polypeptide chain and did not contain sulfhydryl groups or serine residues essential to its activity. The enzyme was activated by Co 2+ and by Mn 2+ , cobaltous ions being much more effective than manganous ions. Its isoelectric point was 5.6 and no evidence of isozymes was seen during isoelectric focusing. Homocarnosinase had a broader specificity, higher solubility, lower stability, and different metal ion sensitivity than hog kidney carnosinase (EC 3.4.13.3). Carnosinase was present in most tissues of the rat, whereas homocarnosinase was detected only in kidney, uterus, lung, and liver.

Cathepsins J and K: High molecular weight cysteine proteinases from human tissues

Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.