Dipeptidyl-aminopeptidase II in human cerebrospinal fluid: changes in patients with Parkinson's disease

By using a sensitive and specific method, DAP II activity was found in CSF. DAP II activity in CSF of control patients without neurological diseases was 0.416 +/- 0.141 (mean +/- SD) nmole/min/ml and was higher than DAP IV activity in CSF, 0.221 +/- 0.062 (mean +/- SD) nmole/min/ml. In contrast, DAP II activity in serum was 1.16 +/- 0.16 (mean +/- SD) nmole/min/ml and was lower than serum DAP IV activity [41.85 +/- 3.36 (mean +/- SD) nmole/min/ml]. This relatively high activity of DAP II in CSF compared with the activity of DAP IV in CSF together with recent histochemical evidence on the localization of DAP II in some neurons (7) suggests that CSF DAP II may be derived from the brain and may be a marker of some peptidergic neurons. DAP II activity in CSF of patients with Parkinson's disease was significantly increased, whereas DAP IV activity in CSF did not change significantly.     

Serum activities of dipeptidyl-aminopeptidase II and dipeptidyl-aminopeptidase IV in tumor-bearing animals and in cancer patients

Serum activities of dipeptidyl-aminopeptidases (DAP) II and IV were measured in tumor-bearing animals and in patients with blood and solid cancers using highly sensitive and specific fluorometric methods. In mice with intraperitoneal or subcutaneous implantation of Ehrlich ascites tumor cells, serum DAP II activity was increased and serum DAP IV activity was decreased, resulting in a significant increase in the ratio of serum DAP II and DAP IV activities. The increase in the ratio of these two activities paralleled the size of the subcutaneous tumors. However, both serum DAP II and DAP IV activities were increased in rats with experimental hepatic cancer induced by 3'-methyl-4-dimethylaminoazobenzene, and the increase in the ratio of the two activities was not significant. In cancer patients, as compared with healthy subjects, serum DAP II activity was increased and serum DAP IV activity was decreased, the ratio of serum DAP II and DAP IV activities being markedly increased in cancer patients. Both serum DAP II and DAP IV activities were increased in patients with hepatic cancer as were those in rats with hepatic cancer, but the increase in DAP II was greater than that of DAP IV; thus the ratio of the two activities increased significantly. These data suggest that the increase of the serum DAP II/DAP IV ratio could be a biochemical index of cancer.     

Cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells

Dipeptidyl aminopeptidase II (DAP II) was demonstrated cytochemically at light and electron microscope levels in rat macrophages and mast cells using Lys-Ala-4-methoxy-2-naphthylamide as a specific substrate. The enzyme which was found to be lysosomal in both cell types, was analyzed biochemically in extracts by measuring fluorometrically the liberated naphthylamine, and was visualized in sections microscopically using azo-coupling methods. DAP II was further characterized by isoelectric focusing techniques. Macrophage DAP II was found to be typical of that found in other rat tissues in terms of its structural latency, substrate specificity, inhibitor sensitivities, and pH activator requirements. Addition DAP II isozymes, not previously recognized, were observed.     

Developmental Pattern for Deoxyhypusine Hydroxylase in Rat Brain

Deoxyhypusine hydroxylase catalyzes the formation of hypusine from deoxyhypusine in a precursor form of eukaryotic initiation factor 4D (eIF-4D). The enzymatic activity was examined in mammalian brain homogenates and the results were consistent with the existence of deoxyhypusine hydroxylase levels comparable to those occurring in other mammalian tissues. Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in cow brain (1.82 units/ mg of protein). In the rat the enzyme was found to be unevenly distributed among various brain regions. The parietal cortex contained the highest specific activity (2.1 units/mg of protein). Rat brain deoxyhypusine hydroxylase was mainly present in the postmicrosomal supernatant (81% of the total activity). The highest specific activity (3 units/mg of protein) was observed in the rat brain during the first few days of life. Thereafter the activity started to decline, and continued to do so for 15 days, remaining throughout the rest of life at levels of less than one-half that of newborn.     

Expression and localization of malate synthase during maturation and dessication of castor bean seeds

Summary Enzymatic levels and subcellular localization of malate synthase in maturing seeds of castor bean (Ricinus communis cv. Hale) are reported. Extracts of maturing seeds exhibited moderately high specific activity (9.68 nmoles/min/mg protein) at 15–20 DAP and lower specific activity (0.49) in mature, dry seeds. Subcellular localization of the enzyme during seed maturation was primarily cytosolic (85%). The remainder of the activity in sucrose gradients was located at high density (1.21 g/cm³). Dry seeds did not contain organelle-bound malate synthase activity. In extracts of 4-day germinated seeds the enzyme was present at high specific activity (12.8 nmoles/min/mg protein) with better than 85% of the total activity in glyoxysomes (1.24 g/cm³).Two polypeptides, 62kDa and 66kDa, reactive with anti-malate synthase were detected at high density in sucrose gradients of homogenates of late-maturing seeds (60 DAP); dry seeds; and seeds imbibed for 6 h. One polypeptide, 62 kDa, in 4-day germinated seeds, reacted with anti-malate synthase. Immunoreactive polypeptides in late-maturing and dry seeds were present at approximately 1/760 of the level found in 4-day germinated seeds. We conclude that malate synthase activity is prominent during early seed maturation but is very low and minimally compartmentalized during late maturation. The rapidly sedimenting immunoreactive polypeptides from dry seeds are enzymatically inactive and are presumed to be of no physiological significance.Abbreviations DAP days after pollination - MS malate synthase - EDTA ethylenediamine tetraacetic acid - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis - BSA bovine serum albumin - IgG gamma globulin     

Pharmacologic properties of a phosphate ester of dopamine

The 3 or 4 phosphate ester of dopamine (PD) was hydrolyzed by homogenates of rat tissues to give inorganic phosphate (Pi) and dopamine. The rate of hydrolysis of PD by kidney homogenates was increased by exogenous MgCl2 but not CaCl2 or KCl. The activity of brain, heart or liver homogenates was insensitive to the added salts. Several lines of evidence indicate that alkaline phosphatase activity contributes to the high rate of PD hydrolysis by the kidney but not brain homogenate. The intravenous infusion of PD at 12 mumole/kg in one hr to anesthetized rats increased the dopamine content of the plasma, kidney and heart without altering brain or liver dopamine. The results suggest that PD may be more effective than dopamine for increasing dopamine levels of the kidney. In addition, the hydrolysis of PD by brain homogenates, which is independent of alkaline phosphatase activity, suggests that specific enzymes exist for the metabolism of PD.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

Altered angiotensin-converting enzyme in lung and extrapulmonary tissues of hypoxia-adapted rats

The effects of exposing rats to hypoxia (10% O2) at normal atmospheric pressure for periods of 14 or 28 days on angiotensin-converting enzyme (ACE) activity and stores of angiotensin I (ANG I) and angiotensin II (ANG II) in lung, kidney, brain, and testis were examined. ACE activity was measured by spectrophotometric assay, and active sites of ACE were estimated by measuring the binding of 125I-351A [N-(1-carbonyl-3-phenyl-propyl)-L-lysyl-L-proline], a highly specific active site-directed inhibitor of ACE, to tissue homogenates and perfused lungs. Hypoxia exposure produced progressive reductions in ACE activity in lung homogenates and in ACE inhibitor binding to perfused lungs. ANG II levels in lungs from hypoxia-adapted animals were significantly less than air controls, suggesting that the reduction in intrapulmonary ACE activity was associated with reduced local generation of ANG II. ACE activity was increased in kidney and unchanged in brain and testis of hypoxia-adapted rats compared with air controls. Thus the effects of chronic hypoxia on catalytically active ACE and ACE active sites in the intact animal were organ specific. Adaptation to chronic hypoxia did not significantly alter plasma renin activity or ANG I or ANG II levels or serum ACE content. The hypoxia-induced alterations in lung and kidney ACE were reversible after return to a normoxic environment.     

An Organoselenium Drug with Antioxidant Activity and Free Radical Scavenging Capacity In Vitro

Organoselenum compounds have been reported to have a wide range of pharmacological properties. Amine-based diselenide, (Z)-N-(4-methylbenzylidene)-1-(2-((2-(1-((E)-4-methyl benzylideneamino)ethyl)phenyl)diselanyl)phenyl)ethanamine ethyl)phenyl) diselanyl) phenyl) ethylimino) methyl)phenol (compound A), and diphenyl diselenide (PhSe)2 were screened for in vitro antioxidant activity. Compound A and (PhSe)2 were tested against sodium nitroprusside (SNP)- and Fe(II)-induced thiobarbituric acid-reactive species (TBARS) in rat brain homogenates. The radical scavenging activity was measured by 1,1-diphenyl-2-picrylhydrazyl assay. Both compounds A and (PhSe)2 decreased Fe(II)- and SNP-stimulated TBARS production in rat brain homogenates. Compound A exhibited the strongest antioxidant activity in the radical scavenging assay, although (PhSe)2, the simplest of the diaryl diselenide, presented no activity. In conclusion, the results of the present investigation indicated that compound A and (PhSe)2 had preventive effects against SNP- and Fe(II)-induced oxidative stress in rat brain homogenates. The amine group in the organic moiety dramatically changed the potency of amine-based diselenide.     

ACTIVITIES OF 3,4-DIHYDROXY-l-PHENYLALANINE AND 5-HYDROXY-l-TRYPTOPHAN DECARBOXYLASES IN RAT BRAIN: ASSAY CHARACTERISTICS AND DISTRIBUTION

Abstract— Optimal assay conditions for decarboxylation of 3,4-dihydroxy- l -phenylalanine (DOPA) and 5-hydroxy- l -tryptophan (5-HTP) were determined in homogenates of rat brain by use of a sensitive, precise microradiometric technique. The two activities exhibited widely different optima for pH, temperature and substrate concentrations. The activity of 5-HTP decarboxylase was stimulated 2-fold by added pyridoxal-5-phosphate and was relatively resistant to antagonists of pyridoxal-P. By contrast, the activity of DOPA decarboxylase was stimulated 20-fold by added coenzyme and could be completely inhibited by carboxyl trapping agents. DOPA decarboxylase activity in subcellular fractions of brain was associated predominately with the soluble fractions and its distribution in the various fractions closely paralleled that of lactic acid dehydrogenase. 5-HTP decarboxylase activity in brain was distributed almost equally between soluble and particulate fractions, and its distribution within the particulate fractions differed from that of succinic acid dehydrogenase. The two decarboxylases in brain exhibited a 7-fold divergence in relative specific activity when their respective distributions in subcellular fractions were compared. Similarly, the regional distributions of the two decarboxylases in rat brain did not parallel one another; e.g. there was a 4-fold difference between the ratio of the two activities in cerebellum and that found in the corpus striatum.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S1/S2 segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 microns. Maximal DAP II activities were demonstrated at pH 5.5. The Km of DAP II was about 2.3 mM. In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S1/S2 segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.     

Uptake of iodinated human kidney alpha-D-mannosidase by rat liver- Association with membrane elements and stability in vivo and in vitro.

1. Human kidney alpha-D-mannosidase (form A) was labelled with 125I to a specific radio-activity of approx. 2250muCi/mg of protein, essentially without loss of enzymic activity. The enzymic activity and radioactivity of the iodinated material also co-migrated in gel filtration and gel electrophoresis. 2. The binding of 125I-labelled mannosidase in vitro to particulate material in liver and kidney homogenates was of the other of 2 pg/mg of particulate material in liver and kidney homogenates was of the order of 2pg/mg of particulate protein withing 16h at 37 degrees C, and essentially zero in intervals of up to 60 min. The degradation in vitro of labelled exogenous mannosidase was of the order of 10-20pg/ 16th per mg of protein in postnuclear supernatant, and it was saturated entirely within 1h at 37 degrees C. 3. The binding of labelled mannosidase in vivo to particulate elements of liver homogenates 60 min after intravenous injection was at least 10 times higher in terms of specific radioactivity than the highest value attainable in vitro. Virtually all exogenous enzyme bound to liver particulate material could be recovered in macromolecular form after disruption of membranes by detergents. 4. The radioactive enzyme bound to liver particulate material could be detached almost completely by shearing, repeated freezing and thawing, and exposure to strong detergents under conditions that do not eliminate rough-endoplasmic-membrane structure. It could bot be released, however, by high salt concentration (0.5M-KC1) or by exposure to weak detergents such as Tween 80. The particle-bound enzyme should thus be associated with plasma membranes and lysosome-like elements. 5. Of the rat tissues studied, only liver could approach, within 60 min after the injection, the concentration of exogenous mannosidase found in the blood serum. The activity per g tissue weight fell progressively from liver (60% of serum value) to kidney (16% of serum value), lung (8% of serum vlaue), spleen (6% of serum value) and brain (0.9% of serum value). Most of the radioactive enzyme found in tissues other than liver appeared to be present in a free form, whereas in liver more than 50% of the labelled enzyme was associated with membrane elements.     

Production of anti-rat islet cell surface antibody in guinea pigs

Anti-rat islet serum was prepared in guinea pigs by multiple subcutaneous inoculations of rat islets homogenates emulsified in complete Freund's adjuvant (CFA). The anti-rat islet serum was cytotoxic against rat spleen cells in the presence of complement and the nonspecific antibodies were observed with homogenates of rat livers and spleens. After absorption, the serum lost the cytotoxicity against the rat spleen cells yet showed specific cytotoxicity against the rat islet cells. The binding capacity of anti-rat islet antibody was determined by the indirect immunofluorescence test using FITC conjugated rabbit anti-guinea pig IgG serum. As the guinea pig anti-rat islet serum contained anti-insulin antibody, the role of this antibody in this cytotoxic activity and surface immunofluorescence was studied. However, the anti-insulin antibody used as the control showed neither cytotoxicity nor surface immunofluorescence. After neutralizing the anti-insulin antibody in the antiserum with insulin, the serum remained cytotoxic to the rat islet cells and a surface immunofluorescence appeared. These data show that specific anti-rat islet cell surface antibody can be produced in guinea pigs by multiple inoculations of rat islets homogenates with CFA.     

Endopeptidases 24.16 and 24.15 Are Responsible for the Degradation of Somatostatin, Neurotensin, and Other Neuropeptides by Cultivated Rat Cortical Astrocytes

Abstract: Several neuropeptides, including neurotensin, somatostatin, bradykinin, angiotensin II, substance P, and luteinizing hormone-releasing hormone but not vasopressin and oxytocin, were actively metabolized through proteolytic degradation by cultivated astrocytes obtained from rat cerebral cortex. Because phenanthroline was an effective degradation inhibitor, metalloproteases were responsible for neuropeptide fragmentation. Neurotensin was cleaved by astrocytes at the Pro¹⁰-Tyr¹¹ and Arg⁸- Arg⁹ bonds, whereas somatostatin was cleaved at the Phe⁶-Phe⁷ and Thr¹⁰-Phe¹¹ bonds. These cleavage sites have been found previously with endopeptidases 24.16 and 24.15 purified from rat brain. Addition of specific inhibitors of these proteases, the dipeptide Pro-He and N -[1-( RS )-carboxy-3-phenylpropyl]-Ala-Ala-Phe-4-aminobenzoate, significantly reduced the generation of the above neuropeptide fragments by astrocytes. The presence of endopeptidases 24.16 and 24.15 in homogenates of astrocytes could also be demonstrated by chromatographic separations of supernatant solubilized cell preparations. Proteolytic activity for neurotensin eluted after both gel and hydroxyapatite chromatography at the same positions as found for purified endopeptidase 24.16 or 24.15. In incubation experiments or in chromatographic separations no phosphoramidon-sensitive endopeptidase 24.11 (enkephalinase) or captopril-sensitive peptidyl dipeptidase A (angiotensin-converting enzyme) could be detected in cultivated astrocytes. Because astrocytes embrace the neuronal synapses where neuropeptides are released, we presume that the endopeptidases 24.16 and 24.15 on astrocytes are strategically located to contribute significantly to the inactivation of neurotensin, somatostatin, and other neuropeptides in the brain.     

Binding of [3H]gamma-vinyl-GABA to GABA-transaminase in brain homogenates

[3H]Gamma-vinyl-GABA, an irreversible inhibitor of GABA-transaminase, was used to label the enzyme in homogenates of rat brain. The binding procedure utilized was found to be specific for GABA-transaminase and linear with tissue obtained from several regions of rat brain up to concentrations of 8 micrograms protein/microliter. The specific binding was directly proportional to the activity of the enzyme measured in vitro and was completely inhibited by the GABA-transaminase inhibitors aminooxyacetic acid (100 microM) and 3-mercaptopropionic acid (1.0mM). The binding procedure was used to estimate the amount of active enzyme present in a homogenate of striatal tissue.     

Udp-galactose: glycoprotein galactosyltransferase activity in a clonal line of rat brain.

1. UDPgalactose:glycoprotein galactosyltransferase (EC 2.4.1.-) activity was demonstrated in homogenates from whole rat brain, isolated neuromal perikarya, enriched glial cell fractions, and cultured rat glial tumor cells (clone C6). 2. Galactosyltransferase activity was enriched 3-9-fold in neuronal perikarya and 1.4--1.8-fold in the glial cell fraction over the activity in whole brains from 19- and 40-day-old rats. The activity of galactosyltransferase in neuronal perikarya decreased with age. Extensive contamination of the glial cell fraction with membranous fragments appeared to obscure the precise specific activity of this fraction. 3. The specific activity of the enzyme in glial tumor cells was 4--8-fold higher than in brain tissue when the enzyme was assayed under identical conditions using endogenous and different exogenous acceptors. 4. Galactosyltransferase activities from adult brain and glial tumor cells had similar properties. They both required Mn-2 plus and Triton, and exhibited pH optima between 5 and 7. The apparent Km of the enzyme for UDPgalactose was 1.3-10-minus 4 M for brain tissue and 2.2-10-minus 4 M for glial tumor cells. 5. The high galactosyltransferase activity in glial tumor cells and in neuronal perikarya of younger rats is compatible with the possibility of a role of this enzyme in developing brain.     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima

For the measurement of the enzymatic activity of GM1-ganglioside (II3 NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl) galactosyl-glucosylceramide) beta-galactosidase in crude enzyme samples, a microassay using nonradioisotopic GM1-ganglioside was devised. To reduce the volume of the reaction mixture and eliminate the interferences due to the fluorescent contaminants in the reaction mixture, NADH, a product after the oxidation of the released galactose with NAD and beta-galactose dehydrogenase, was fluorometrically estimated by use of high-performance liquid chromatography. By this method, as little as 10 pmol of galactose can be detected. Using rat brain homogenates as an enzyme sample, the several parameters were reexamined to define the optimal conditions for the assay. This assay method was also applied to human cultured skin fibroblast homogenates, and it was found that this method can be used for the diagnosis of GM1-gangliosidosis, instead of the usual method using the radioisotope-labeled natural substrate.     

Glycolytic enzyme levels in synaptosomes

The specific activities of glucosephosphate isomerase, aldolase, triosephosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, pyruvate kinase and lactate dehydrogenase were all higher in the synaptoplasmic fraction from rat brain than in 100,000 g supernatant fraction of rat brain homogenates when the supernatants were prepared in high ionic strength solutions. Four enzymes in synaptosomes and two enzymes in homogenates were associated with particulate fractions as indicated by the large increase in specific activity of the enzymes when samples were treated with 0.3 M KCl before centrifugation. Glucosephosphate isomerase, aldolase, pyruvate kinase and lactate dehydrogenase were the enzymes that showed a large increase in specific activity following salt treatment of isolated, synaptosomal membrane while aldolase and pyruvate kinase were the two enzymes which showed a large increase in specific activity in the high speed supernatant fractions. Because the specific activities of many enzymes are found to be elevated not only in synaptosomes but in synaptosomal membrane fractions it is suggested that these enzymes may provide the potential for significantly enhanced glycolysis at these locations.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.